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Pneumonia and skin diseases are some types of severe infections occur worldwide due to pathogenic bacteria such as Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). These bacteria’s do contribute towards other infections in human and thus lead towards unhealthy life. The aim of this study was to determine the fatty acid composition and to investigate the toxic effect of seed oil of Neem, genus Azadirachta of Azadirachta indica (Family: Meliaceae) against two multi-drug resistant bacteria, namely; E. coli and S. aureus. The oil was extracted for 6 h through soxhlet method using hexane as solvent. The fatty acid composition was analyzed via Gas Chromatography-Mass Spectrometry (GC-MS). The composition resulted in detection of 8 fatty acids, whereby the dominant compound is linoleic acid of 34.69% and followed by oleic-, stearic-, palmitic-, arachidic-, behenic-, lignoceric-, and palmiticoleic acid at the percentage of 20.46, 20.42, 18.66, 3.59, 0.80, 0.55, and 0.17%, respectively. The bioassays were conducted through in vitro activity via disc diffusion method with five (5, 20, 50, 80, and 100 mg/mL) different concentrations. Streptomycin and 1% Dimethyl sulfoxide (DMSO) were used as positive and negative controls respectively. The bioassay provided inhibition zone that lies between 8.7 to 11.7 mm and 8.7 to 13.0 mm for E. coli and S. aureus respectively at the concentration of 5 to 100 mg/mL. The antibacterial activity of Neem (A indica) seed oil against these bacteria depends on the correlation of its concentration and thus isolation of bioactive components should be conducted.