The present study was carried out to investigate the chemical constituents of Ziziphus spina-christi seed oil and to assess its potential antimicrobial activity. GC-MS analysis revealed 21 constituents. Major constituents are: 9-octadecenoic acid methyl ester (30.81%), 9, 12-octadecadienoic acid methyl ester (21.28%), hexadecanoic acid methyl ester (12.23%) and Methyl stearate (10.29%). Ziziphus spina-christi seed oil was evaluated for antimicrobial activity via cup plate agar diffusion bioassay against six standard bacterial strains: (Gram positive: Staphylococcus aureus and Bacillus subtilis; Gram negative: Escherichia coli and Pseudomonasa aeruginosa and the fungi Candida albicans and Aspergillus niger). The oil gave excellent activity against the bacterial strains Escherichia coli and Bacillus subtilis at 100mg/ml. It also showed excellent activity against Bacillus subtilis at 50mg/ml. Good activity was observed for Escherichia coli at the concentrations: 50, 25 and 12.5mg/ml.