The use of anti-fungal agents has increased dramatically in recent years and new drugs have been developed. Several methods are available for determinations of their specific biological activities, i.e. the standard method for minimum inhibitory concentration-determination is described in M-38 [Clinical and Laboratory Standards Institute document M-38 (CLSI M-38)]. However, alternative methods, such as the E-test, are currently available in Mycology laboratories. The susceptibilities of clinical isolates of Aspergillus spp. (n = 29), Fusarium spp. (n = 5), zygomycetes (n = 21) and Schizophyllum (n = 1) were determined for itraconazole, voriconazole and posaconazole, using the CLSI M-38-A broth dilution method and also by the E-test. A good overall agreement (83.7%) between the two methods for all drugs and organisms was observed. Analyses of voriconazole showed a better agreement (93%) between the methods than posaconazole and itraconazole (85% and 74% respectively). Aspergillus spp. were the most susceptible fungi to the anti-fungal agents tested in this study. Posaconazole was the most active drug against filamentous fungi in vitro, followed by itraconazole and voriconazole. The latter (voriconazole) demonstrated no significant in vitro activity against zygomycetes.