Oil analysis and antimicrobial activity from seeds of elite genotype of Pongamia pinnata was carried out in the current study. The highest oil yield (33%) from seeds was recovered in n-Hexane. Physico-chemical properties of crude oil established suitability of P. pinnata for its use as a potential biofuel crop. The total mono unsaturated fatty acid (oleic acid 46%) present in seed oil was more in comparison to polyunsaturated fatty acid (33%) as analyzed by GC-MS. Seed oil also showed inhibition against the tested fungal and bacterial cultures. However, the efficacy of antimicrobial activity of the seed oil at four concentration levels (50%, 80%, 90% and 100%) against various pathogenic indicators was found to be concentration-dependent. The obtained results confirmed the use of seed oil from well characterized elite genotype of Pongamia as diesel fuel and in pharmaceuticals. (C) 2009 Elsevier Ltd. All rights reserved.