The study of antibacterial activity of oil from Tamarindus indica Linn seed and fatty acids was conducted in this research. Oil of this plant seed was isolated by extraction and fractionation. Constituents of fatty acid in the oil identified as methyl ester fatty acids. The methyl ester fatty acid was carried out by trans-esterification with methanol/BF3. The methyl ester was identified by gas chromatography and mass spectrometer (GC-MS). There are 13 fatty acids in the Tamarindus indica seed oil including octanoic, decanoic, dodecanoic, tetradecanoic, hexadecanoic, octadecanoic, eicosanoic, docosanoic, tetracosanoic, 11-octadecenoic, 11-eicosenoic, 9,12-octadecadienoic, and 9-octadecenoic acids. The transformation of tamarind seed oil into fatty acids was carried out through hydrolysis with potassium hydroxide solution followed by acidification with a hydrochloric acid solution. The physical and biological properties of the seed oil and fatty acids against antibacterial activity are reported in this paper.