Infections caused by Candida species (candidosis) have greatly increased over recent years, mainly due to the escalation of the AIDS epidemic, population ageing, increasing number of immunocompromised patients and the more widespread use of indwelling medical devices. Candida albicans is the main cause of candidosis, however, non-Candida albicans Candida (NCAC) species such as Candida glabrata, Candida tropicalis and Candida parapsilosis are now frequently identified as potential human pathogens. This increased incidence of NCAC in human infection can be attributed to improved identification methods but might also be a reflection of the high level of resistance often exhibited by these species to certain antifungal agents. Candida species pathogenicity is facilitated by a number of virulence factors, most importantly adherence to medical devices and/or host cells, biofilm formation, and secretion of enzymes, such as proteases. Despite intensive research to identify pathogenic factors in fungi, particularly in C. albicans, relatively little is known about the virulence determinants of NCAC species. Thus, the work described in this thesis examined several of the most relevant virulence factors (adhesion, biofilm formation ability, tissue colonisation and invasion, and expression of hydrolytic enzymes) of clinical isolates of C. glabrata, C. tropicalis and C. parapsilosis recovered from different body sites (oral cavity and urinary and vaginal tracts). The study of NCAC biofilms, in terms of formation, structure, matrix composition, and metabolic activity, was the first goal of this research. Total biomass quantification, showed that all NCAC strains were able to form biofilms, although this was less extensive for C. glabrata compared to C. parapsilosis and C. tropicalis. Scanning electron microscopy (SEM) revealed structural differences for biofilms with respect to cell morphology and spatial arrangement. Furthermore, C. parapsilosis matrices had large amounts of carbohydrates and low protein. Conversely, matrices extracted from C. tropicalis biofilms had low amounts of carbohydrates and protein. Interestingly, the composition of C. glabrata biofilm matrices' was high in both protein and carbohydrate contents. It was also evident that there were intrinsic differences in terms of metabolic activity amongst biofilms of NCAC species, and no correlation was found concerning colony forming units (CFUs) and biofilm metabolic activities determined by XTT reduction. Another objective of the work described was to study the adhesion and biofilm formation ability of several clinical urinary isolates on to silicone in the presence of artificial urine (AU) and the role of Candida surface properties (hydrophobicity and zeta potential) in these events. Silicone colonization by NCAC species in the presence of AU showed that, all urinary isolates were able to adhere to silicone, but in a species and strain dependent manner. However, these differences in adhesion could not be correlated with cell surface properties (hydrophobicity and zeta potential). Moreover, despite the high number of cultivable cells biofilms were not observed and confocal scanning laser microscopy (CLSM) showed an absence of extracellular polymeric material for all strains. The pathogenesis of C. parapsilosis and C. tropicalis was investigated using a commercially available reconstituted human oral epithelium (RHOE), in conjunction with CLSM observation and lactate dehydrogenase (LDH) determination. Furthermore, the role of secreted aspartly proteinases (Saps) on invasion and damage of RHOE was evaluated by real-time polymerase chain reaction (PCR). It was possible to observe that all C. tropicalis and C. parapsilosis strains were able to colonize the tissue, however, this was in a species- and straindependent manner. Candida parapsilosis revealed low invasiveness after 12 h of infection and extensive damage was evident after 24 h when assessed using histological examination and LDH determination. Moreover, C. tropicalis was found to be highly invasive after 12 h of infection, with extensive tissue damage occurring also after 24h. Real time-PCR of SAP genes showed that expression was strain dependent for both species. Furthermore, the results suggested that the proteinases were not involved in invasion of RHOE by C. tropicalis and C. parapsilosis, but indicated a role for these enzymes in tissue damage caused by C. parapsilosis. Finally, single and mixed infection of RHOE by C. glabrata or/and C. albicans were examined, using CLSM and peptide nucleic probes by in situ hybridization (PNA FISH). It was found that the invasiveness of C. glabrata strains was enhanced in the presence of C. albicans. In summary, this work underlines both species and strain differences in terms of virulence factors associated with C. glabrata, C. parapsilosis and C. tropicalis. Furthermore, there was clear evidence demonstrating the importance of the use of new techniques including CLSM and molecular analysis tools enabling the elucidation of the mechanisms of virulence. By increasing our knowledge on Candida pathogenesis, potential therapeutic targets may well be identified that can be used as adjuvant for new therapies.