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AIMS This study evaluated the antifungal, antibiofilm, and molecular docking of 2-Chloro-N-phenylacetamide against clinical isolates of Candida tropicalis and Candida parapsilosis. METHODS AND RESULTS MIC of the test drugs was determined by microdilution. A1Cl obtained MIC values ranging from 16 and 256 μg/mL. Fluconazole MIC ranging from 16 and 512 μg/mL. MIC of A1Cl showed fungicide activity, emphasizing the solid antifungal potential of this drug. An association study was performed with A1Cl and fluconazole (checkerboard), revealing indifference by decreasing. Thus, we conducted this study using A1Cl isolated. In the micromorphological assay, the test drugs reduced the production of virulence structures compared to the control (concentration-dependent effect). A1Cl inhibited in vitro biofilm formation at all concentrations tested (1/4MIC to 8xMIC) (p<0.05) and reduced mature biofilm biomass (p<0.05) against C. tropicalis and C. parapsilosis. In the ex vivo biofilm susceptibility testing (human nails fragments), A1Cl inhibited biofilm formation and reduced mature biofilm biomass (p<0.05) more than 50% at MIC. Fluconazole had a similar effect at 4xMIC. In silico studies suggest that the mechanism of antifungal activity of A1Cl involves the inhibition of the enzyme dihydrofolate reductase rather than geranylgeranyltransferase-I. CONCLUSIONS The results suggest that A1Cl is a promising antifungal agent. Furthermore, this activity is related to attenuation of expression of virulence factors and antibiofilm effects against C. tropicalis and C. parapsilosis. SIGNIFICANCE AND IMPACT OF THE STUDY Our study provides the first evidence that A1Cl, a novel synthetic drug, has fungicidal effects against C. tropicalis and C. parapsilosis. Furthermore, in vitro and ex vivo biofilms assays have demonstrated the potential antibiofilm of A1Cl. The mechanism of action involves inhibiting the enzyme dihydrofolate reductase, which was supported by in silico analyses. Therefore, this potential can be explored as a therapeutic alternative for onychomycosis and, at the same time, contribute to decreasing the resistance of clinical isolates of C. tropicalis and C. parapsilosis.