Objective: To isolate, purify, and characterize gossypol from the fruits of Thespesia populnea (L) Sol. ex Correa, test its anti-dermatophytic activity, identify its targets on the dermatophyte, and confirm the binding of gossypol with the fungal target by molecular docking study. Methods: Gossypol from Thespesia populnea was characterized by high performance liquid chromatography, liquid chromatograph-mass spectrometry, Fourier transform infrared spectroscopy, and nuclear magnetic resonance. The anti-dermatophytic activity of gossypol was tested against four different dermatophytes, viz. Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum canis, and Microsporum gypseum. Trichophyton mentagrophytes was selected for further studies. The inhibitory mode of action of gossypol on Trichophyton mentagrophytes was determined by analyzing the modulation of gene expression in various pathways of the dermatophyte. Results: Gossypol inhibited all the dermatophytes. The minimum inhibitory concentrations were 12.5 μg/mL for Trichophyton mentagrophytes and Microsporum canis and 25 μg/mL for Trichophyton rubrum and Microsporum gypseum. The minimum fungicidal concentrations were 50 μg/mL for Trichophyton mentagrophytes, 100 μg/mL for Microsporum canis and Trichophyton rubrum, and 200 μg/mL for Microsporum gypseum. Gossypol inhibited the mRNA expression of metalloprotease (MEP4) and isocitrate lyase (ICL). The binding of gossypol with the enzymes was confirmed by molecular docking studies. The best docking poses were found and the low binding energies were recorded with the two target enzymes. Conclusions: Gossypol is a potential antifungal agent and can be further explored as an anti-dermatophytic drug.