Lipids possess versatile biological properties in human health and nutrition. The compositions of lipids were investigated on the raw, boiled and fermented seeds of Pentaclethra macrophylla n-hexane extracts. The seed extracts were esterified, and chemical composition of the fatty acids was evaluated using gas chromatography-mass spectrometry (GC-MS). Subsequent chromatographic purification and isolation on the lipids was carried out using column chromatography and thin layer chromatographic techniques. The antimicrobial activity of extracts was evaluated using disc diffusion and broth dilution techniques on selected bacterial and fungal species, Methicillin resistant Staphylococcus aureus (MRSA), Staphylococcus aureus, Shigella dysenterea, Salmonella typhi, Proteus mirabilis, Corynebacterium ulcerans, Escherichia coli, Candida krusei, Candida albicans and Candida tropicalis. GC-MS analysis revealed that fatty acids such as linoleic acid, butyl 9,12- octadecadienoate acid, oleic acid, hexadecanoic acid, methyl 20-methyl heneicosanoate, tetracosanoate acid, ethyl tetracosanoate and methyl stearate were common in the raw, boiled and fermented seed extracts. However, linoleic acid content was higher (60%) in fermented seeds, indicating an increased production due to fermentation effect. The extracts have demonstrated growth inhibition on bacterial and fungal species with broad-spectrum activity for the fermented seeds (17-19 mm; MIC 5 mg/mL) and the raw and boiled seeds (16-18 mm; MIC 5 mg/mL). Monoglycerides were isolated and structures elucidated using H-NMR and C-DEPT as 2,3-dihydroxypropyl tetracosanoate (1), 2,3-dihydroxypropyl pentacosanoate (2) and 2,3- dihydroxypropyl hentriacontylate (3). Compounds (2) and (3) are reported for the first time from the seeds. This report indicated the potential benefit of P. macrophylla seeds fermentation to human nutrition and health.