Ocimum basilicum has been widely used in traditional medicine. Rural communities have used fixed oils for variety purposes since a long time ago. They used for cosmetic applications, fuel, medicine and food. The aim of this study was to characterize the physicochemical properties and fatty acid composition of O. basilicum seed oil. Lipids were determined by continuous extraction in a Soxhlet apparatus for 6 hours using hexane as solvent. The physicochemical properties of the oil were assessed by standard and established methods. The fatty acids composition of the seed oil was determined by GC-MS. The Pale yellow with camphor odor oil extracted from the seed has the following properties: yield, 18.01%; freezing point, -2°C; melting point, 5°C; boiling point, 215°C; refractive index (25°C), 1.48532; iodine value, 108.6 g/100 g of oil; peroxide value, 4.6 meq. O2/kg of oil; free fatty acids, 0.20%; acid value, 4.0 mg of KOH/g of oil; saponification value, 164.2 mg KOH/g of oil; unsaponifiable matter, 1.6; moisture and volatile value, 4.97 (wt%); density, 0.91372 g/cm3; viscosity, 10.29 mm2/s; specific gravity, 0.9210. Fatty acids composition showed that linolenic- (43.92%) was the major fatty acid and followed by linoleic- (32.18%), palmitic- (13.38%), stearic- (6.55%), palmitoleic- (0.78%), arachidic- (0.72%), anteisomargaric- (0.45%), nonadecylic- (0.28%), gondoic- (0.27%), margaric- (0.20%), behenic- (0.17%), heneicosylic- (0.14%), lignoceric- (0.13%) and myristic acid (0.11%). Therefore, recommended that more and advanced investigations should be undertaken for this abundant oil as natural source for many industrial applications, especially, for applications that require acids like linolenic and linoleic.