Genetic variation of two Ziziphus spina-christi L. (Sidr) varieties was determined by Random Amplified Polymorphic DNA (RAPD). The activity of antioxidant enzymes (peroxidase and catalase) was also determined for the two varieties (Balady and Pakistani). Moreover, the antibacterial effects of different concentrations of aqueous and ethanolic extracts of leaves of the two varieties of Ziziphus spina-christi L. were assessed against Gram-positive bacteria (Streptococcus pyogenes and Staphylococcus aureus) and Gram-negative (Klebsiella pneumoniae, Proteus mirablis and Escherichia coli). The similarity of RAPD profiles was found to be 85%. Our results suggest that Ziziphus spina-christi is an effective source of antioxidants and its leaf extract is a potential promising alternative antibacterial agent against different pathogenic bacteria.