The aim of this study was to determine the antibacterial activity of Metformin against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Antibacterial activity was evaluated using the Kirby Bauer technique. Strains of Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), and Pseudomonas aeruginosa (ATCC 27853) were used, which were exposed to Metformin at a concentration of 250 mg and 500 mg, Ciprofloxacin (CIP) 5 µg, Imipenem (IPM) 10 µg, and Cefoxitin (FOX) 30 µg. Regarding Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, an inhibition halo of 6 mm was presented for Metformin 250 mg, 6 mm. for Metformin 500 mg, and an inhibition halo> 25 mm. with the use of Ciprofloxacin 5 µg, Cefoxitin 30 µg, and Imipenem 10 µg respectively. In conclusion, in vitro, Metformin at doses of 250 and 500 mg had no antibacterial effect against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa.