Juglans regia, commonly known as English or Persian walnut is a deciduous tree with high nutritional, medicinal as well as economical value. To our knowledge, the medicinal importance of J. regia as anti-inflammatory, antibacterial and anti-fungal is not fully exploited. Therefore, the current study focused on the antimicrobial potential of the J. regia’s leaf extracts against several bacteria and fungi such as Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Pseudomonas aeruginosa (multi drug resistant), Klebsiella pneumonia, Klebsiella pneumonia (multi drug resistant), Escherichia coli and Aspergillus Flavus, Fusarium, Penicillium. We evaluated the antibacterial and anti-fungal effects of the leaf extracts using soxhlet extraction in ethanol, methanol and water as solvents and water bath extraction, ethanol, and methanol water extraction. The data revealed a statistically significant (P <0.05) minimum inhibitory zone against S.aureus, E.coli, MRSA and Candida albicans when tested with the ethanol (soxhlet extraction), methanol (soxhlet extraction) and ethanol (water extraction) leaf extracts as compared to the negative controls. Furthermore, the results also indicated a statistically significant (P <0.05) minimum inhibitory zone against fungi like Aspergillus Flavus, Fusarium, Penicillium evaluated with the ethanol, methanol (soxhlet extraction) and ethanol and methanol (water bath extraction) leaf extracts in comparison to the negative controls. In summary, our investigations demonstrated the potential of the J. regia’s leaf extracts as antimicrobial agents against disease causing bacteria and fungi.