An increase in infections due to non albicans species of Candida has been observed in the recent years. The aims of this study were to determine the antifungal activity of 2,4-dihydroxy-N-(3-thioxo-3H-l1,2,4-dithiazol-5-yl)benzenecarbothioamide (DNTDB) against C. albicans, non-C. albicans, dermatophytes, and molds and to evaluate the enzymatic activity of C. albicans strains. We used reference strans C. albicans 10231 ATCC, 200 C. albicans strains, 100 non-C. albicans, 19 dermatophyte strains, and 21 mold strains isolated from different ontocenoses from patients. DNTDB revealed a mean minimum inhibitory concentration (MIC) of 12.5 pg/mL against the reference C. albicans 10231 ATCC strain on Sabouraud agar (SA) and of 6.5 pg/mL on Roswell Park Memorial Institute (RPMI) medium. The mean MIC for C. albicans isolates was of 22.01 ± 7.5 μg/mL on SA, 17.8 ± 7.4 μg/mL on yeast nitrogen base (YNB), and 16.9 ± 7.9 μg/mL on RPMI medium. The mean MIC for non-C. albicans isolates was of 22.4 ± 12.4 μg/mL on SA, 18.2 ± 8.6 μg/mL on YNB and 15.2 ± 9.03 sg/mL on RPMI. Against Trichophyton mentagrophytes v. granulosum, the mean MIC was 10.9 ± 2.04 μg/mL after 5 days of incubation and 21.9 ± 3.8 μg/mL after 15 days, while Trichophyton mentagrophytes . inteiligitale showed a mean MIC of 13.3 ± 5.5 μg/mL and of 20.3 ± 6.1. μg/mL after the same incubation periods, respectively. DNTDB manifested a MIC over the test range of 25-100 μg/mL for molds after 5 days of incubation and inhibited the enzymatic activity of Candida strains. It seems, the new DNTDB demonstrates potential antifungal activity against yeast-like fungus strains, dermatophytes, and molds in vitro.