Increase in resistance among fungal species, to routinely used antifungal agents has necessitated the quest for new drugs. Few studies have revealed that cow's urine can suppress the growth of pathogenic fungi. However, there is limited published report on antifungal effects of formulated cow's urine on fungal isolates. The present study aims at exploring the antifungal potential of cow's urine on various fungal isolates. In this in-vitro experimental study five fungal genera were tested for their susceptibility to amphotericin B, fluconazole and voriconazole and cow urine by well diffusion and point inoculation method. Detection of inhibitory efficacy of cow's urine for the fungal isolates was done by agar dilution method using 5–20% concentration of cow's urine. The environmental isolates of 5 genera: Aspergillus niger, Candida albicans, Penicillium spp., Fusarium spp. and Rhizopus spp. were tested against cow urine for their antifungal susceptibility. The results revealed that the effectiveness was found highest against the fungal spp. of Candida spp. (90%) and Penicillium spp. (78%) followed by Aspergillus niger (70%), Fusarium spp. (65%) and Rhizobium spp. (60%). It was found that the isolates were less sensitive to Itraconazole and fluconazole while found highly susceptible to cow urine treatment. The study concludes that Cow's urine distillate has effective inhibition on fungal species and with a few modifications this can be a good natural alternative for reducing several diseases