This review provides the background for understanding the role of a battery of diagnostic methods in paracoccidioidomycosis (PCM). This systemic mycosis is a disease endemic in many regions of Latin America, with sporadic cases also occurring throughout the world (mycosis of importation). Although excellent laboratory methods for diagnosis are available, there are deficiencies that must be met by continued research. Understanding the uses and limitations of a battery of laboratory methods is essential to diagnose PCM. Clinicians and laboratory directors must be familiar with the uses and limitations of a battery of serologic and mycological tests to accurately diagnose of PCM. Antibody and antigen detections are valuable adjuncts to histopathology and culture. More recently, the gp43 and gp70 antigen detection assay have improved the methodology of diagnosis of this mycosis, which improves reproducibility and facilitates monitoring antigen clearance during antifungal treatment. Furthermore, detection of antigen in cerebrospinal fluid and in bronchoalveolar lavage fluid increases the sensitivity for diagnosis of PCM in central nervous system and in pulmonary infections, respectively.