Essential oil obtained from aerial parts of Ocimum gratissimum collected from Chamba (Tehri Garhwal), Uttrakhand, India by hydrodistillation, was analyzed by GC and GC-MS for its chemical profile, and evaluated for antifungal activity. The analysis revealed the presence of 35 compounds which constitutes 96.27% of the total oil. The major components of the oil were found to be e (32.86%), -terpenolene (22.83%), cis methylbicyclo (4,3,0) non-3-ene (9.08%), camphene (2.65%), -phellandrene (2.12%), -pinene(1.56%), -bourbonene (1.46%), -pinene (1.36%), carophyllene (1.32%), dodecatetraene (1.23%), 3 cyclohexen-1-ol (1.15%) and tert (1.02%),. The essential oil exhibited good antimicrobial activity against few human pathogenic fungi.