This research was carried out to evaluate the antibacterial activity of lyophilized Citrus limon on multi-drug resistant Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhi, Macrococcus luteus and Clostridium difficile. Phytochemical constituent of the lyophilized citrus limon was determined using standard laboratory methods, while the agar well diffusion technique was used to evaluate the antibacterial potential of the lyophilized extract. Phytochemistry of the lyophilized Citrus limon extract revealed the presence of saponins, tannin, flavonoid, glycoside, cardiac, terpenes, anthraquinone, resins phenol, alkaloid, volatile oils and steroid. Lyophilized Citrus limon juice extract had a significant activity (p<0.05) with zone of inhibition as follows 26.00±1.4, 21.00±1.41, 24.50±0.70, 16.50±0.70, 29.50±2.12 and 20.50±0.00 mm at 40 mg/mL against Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Salmonella typhi, Macrococcus luteus and Clostridium difficile, respectively. The minimum inhibitory concentration ranges from 10-80 mg/mL and the minimum bacteriocidal concentration ranges from 80-160 mg/mL. This study provides insight on the antibacterial potential of Citrus limon juice extract against multidrug resistant bacterial pathogens. It is recommended that further studies should be conducted to explore the Citrus limon bioactive compound in the development of therapeutic drug.