Objectives: The application of molecular methods for the laboratory diagnosis of malaria and leishmaniasis was evaluated in our laboratory as compared to conventional methods. Methods: Malaria: Blood samples (1,467) from 928 patients with clinical suspicion of imported malaria were subjected to thin-film microscopy (acridine orange and Giemsa stain) and to different 18SrDNA PCRs alternatively used during the period 2000–2010, including nested- and real-time PCR assays. Leishmaniasis: Different samples (27 bone marrow, 13 cutaneous and 1 splenic biopsies) from 36 patients with clinical suspicion of leishmaniasis were subjected to conventional methods (microscopy and culture) and a 18S-rDNA real-time PCR assay, during 2006–2010. Results: Malaria: By microscopy 208 cases of malaria were diagnosed [173 Plasmodium falciparum (Pf) (83.2%), 11 P. ovale (Po) (5.3%), 12 P. vivax (Pv) (5.8%), 9 P. species (4.3%) and 3 mixed infection (1.4%)], whilst 215 were diagnosed by PCRs [174 Pf (80.9%), 21 Po (9.8%), 8 Pv (3.7%), 3 P. malariae (1.4%) and 9 mixed infections (4.2%)]. Leishmaniasis: A total of 2 cases of leishmaniasis (1 cutaneous and 1 visceral) were diagnosed by both conventional methods and PCR. Regarding the cutaneous case, 2 biopsies were collected including 1 negative by conventional methods. Conclusion: Malaria: Despite microscopy remains the reference diagnostic method (rapid and inexpensive), in some cases molecular assays are the only ones allowing a correct diagnosis of malaria, particularly to detect infections by species other than Pf and mixed infections. PCR proved to be more sensitive and specific than microscopy and changed malaria epidemiology in our area detecting 7 single and 6 mixed infections missed by microscopy, revealing 5 single and 2 mixed infections incorrectly diagnosed by microscopy and giving speciation in 9 cases in which microscopy had limited the result to genus identification. Then, the association of microscopic and molecular assays demonstrated to be essential for an accurate diagnosis of malaria and to administer a targeted therapy. Leishmaniasis: The application of the real-time PCR did not lead in our study to a dramatic improvement in the accuracy of the diagnosis of leishmaniasis, even if the number of the analysed samples was limited. However, real-time PCR is very rapid and specific and it could be successfully associated to the conventional methods in particular in order to improve the diagnosis of visceral leishmaniasis.