OBJECTIVE To determine the association of Candida phenotypes, virulence factors, antifungal sensitivity and clinical response to Fluconazole in Oral leukoplakia (OL). METHODS Sterile swabs were obtained from oral lesions in immunocompetent subjects [30 Homogenous (HOL), 31 Non- Homogenous (NHOL] and normal buccal mucosa in 30 age and sex-matched healthy controls (C). Candida phenotypes, virulence factors (Secreted Aspartyl Proteinase (SAP), Phospholipase (PL), Biofilm formation (BF) and antifungal sensitivity were determined. Clinical features (Size, Erythema, thickness, oral burning sensation (VAS scores) before and after Fluconazole therapy in OL were recorded by two calibrated observers. RESULTS Candida was associated with OL (p ​< ​0.01). Candida albicans was the most common phenotype sensitive to Fluconazole. SAP, PL and BF activity was significantly high in NHOL. Strong positive correlation was seen between SAP, and PL activity and pre-treatment VAS scores in NHOL. There was significant reduction in VAS scores, size of lesion [HOL (p ​< ​0.001) NHOL (p ​< ​0.05)], erythematous areas (67.8%) in NHOL and thickness of lesions (42.6%) in both types OL after Fluconazole therapy with substantial inter-observer agreement. Non albicans candida (NAC) species had similar virulence profiles but resistant to Fluconazole and showed minimal clinical improvement. CONCLUSIONS Virulence activity of Candida in OL increases severity of lesions. Fluconazole is effective against virulent Candida albicans, causes clinical improvement and down-staging from high -risk NHOL to low-risk HOL which can reduce risk of malignant transformation. Detection of highly virulent NAC infection and antifungal sensitivity is recommended in OL recalcitrant to Fluconazole therapy.