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Antibacterial and Anti-inflammatory activities of methanolic extract of Juglans regia L. leaves (Walnut) grown in Libya were investiagted. Hot extraction method by methanol using Soxhlet apparatus was utilized. The antimicrobial effect was studied using agar diffusion method. Human red blood cell membrane stabilization (HRBC) method was used to estimate the anti-inflammatory properties of the leave extract. The extract of leaves exhibited antimicrobial activity against Staphylococcus aureus, and Candida albicans with a minimum inhibitory concentration (MIC) of 25 and 50 mg/ml, respectively. In addition, the extract concentrations of 20, 40, and 100 μg/mL inhibited lysis of HRBC with values of 25.8%, 35.5%, and 70%, respectively. The results showed that the methanolic extract of Juglans regia leaves exhibited antibacterial activity against S. aureus, and antifungal activity against C. albican, and produced anti-inflammatory activity.