Abstract The seed of Pentaclethra macrophylla benth are seen plentifully during the plants’ seasonal fruiting. Though the nutritional value of this seeds had been analyzed, the seed oil had not been characterized to see if it should be of high industrial importance. In this work, the author tried to extract and characterize P. macrophylla seeds oil. Again the anti-microbial screening of the oil is investigated on six pathogenic micro-organisms to know if the natural oil is medicinal or not. The six micro-organisms are: Bacillus cereus, Bacillus lichemiformis, Lactobacillus specie, Escherichia coli, Candida albican and Aspergillus niger. The percentage yield of the oil is 35.08%. Characterization of the oil shows that the acid value is 1.23 mg/KOH g-1, peroxide value is 16 meq, lodine value is 116 mg/100g and saponification value is 207.54 mg/100g. The antimicrobial screening carried out shows that the 0.1ml of P. macrophylla seed oil can inhibit the growth of five out of the six test micro-organisms. The five micro-organisms are: B. cereus, B. lichemiformis, L. species, E. coli and C. albican. At 0.1 ml volume, the oil cannot inhibit the growth of A. niger. The pharmaceutical screening of P. macrophylla benth seed oil confirmed the medicinal value of the oil.