ObjectiveTo study the in vitro antifungal activity of novel 2,5-disubstituted 1,3,4-oxadiazoles. MethodsSixteen novel 2,5-disubstituted 1,3,4-oxadiazoles compounds have been designed, synthesized and characterized by 1HNMR, LC-MS spectra. Their antifungal activities were evaluated with six tested pathogenic fungi in vitro. ResultsAll the title compounds exhibited potent antifungal activities against Candida albicans, Cryptococcus neoformans, Candida parapsilosis, Candida tropicalis, and Trichophyton rubrum, but not Aspergillus fumigatus. The activities of compound 14, 17 and 18 against Candida atbicans and Cryptococcus neoformans (with the MIC80 0.25 μg/ml) were similar to that of fluconazole. The activities of compound 14 and 17 against Trichophyton rubrum (with the MIC80 0.25 μg/ml) were 2 times as high as that of fluconazole and were similar to that of ketoconazole. ConclusionThe 1,2,3-triazole can be efficiently introduced to 1,3,4-oxadiazoles compounds by intermolecular 1,3-dipolar cycloaddition. All the title compounds exhibit certain antifungal activities with broad spectrum. The substituent of the benzene plays an important role in improving the antifungal activity of the compound.