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Background: Many fungal diseases are becoming increasingly resistant to antibiotics, necessitating the development of new or repurposed treatment techniques. Gentian violet (GV) is considered as a triarylmethane stain that is utilized as a histology dye, in Gram's procedure of bacterial detection, and for its antibiotic effectiveness against specific diseases, particularly dermatological infections. At present time, there are just a few research on the antimicrobial properties of gentian violet. Objective: With growing prevalence of multi-resistant drugs, the objective of this work to evaluate the antifungal efficacy of GV against Candida albicans. Methods: Gentian violet has been prepared. The antifungal activities were studied by using well-diffusion method against 10 clinical isolates. C. albicans were diagnosed by using VITEK 2 automated system. Fluconazole used as control agent because it’s high sensitivity to these fungi. Results: Gentian violet dye showed full sensitivity 100% to all C. albicans isolates with mean 26 mm (24-28 mm). Fluconazole was sensitive in 80% of isolates with mean 19.5 mm (10-24 mm). Significant differences have observed between gentian violet and fluconazole (P= 0.000). 40% of specimens isolated from oral cavity, 40% were collected from vaginal swab, and 20% isolates collected from urinary tract. Conclusions: GV is a low cost dye and good to be tolerated topical agent with the potential for utilizing applications in the dermatological branch. Gentian violet showed good antifungal activity against Candida albicans that can be used to treat infection due to these fungal pathogens.