The application of antibiotics to treat bacterial infections poses risks to fish and the ecosystem, highlighting the need for safe, natural alternatives for fish disease treatment. This research focuses on evaluating the antibacterial properties of Melicope ptelefolia extract against various pathogens, including Aeromonas salmonicida, Edwarsiella tarda, Edwarsiella ictaluri, Aeromonas hydrophila, and Pseudomonas aeruginosa. The extract underwent phytochemical analysis, which confirmed the presence of phenolic compounds, flavonoids, saponins, and terpenoids. Antibacterial effectiveness was assessed using the agar diffusion method at 100, 200, 300, 400, and 500 mg/mL concentrations. The results showed that M. ptelefolia extract significantly inhibited the growth of A. salmonicida (13.5 mm to 14.8 mm), E. tarda (12.0 mm to 13.1 mm), E. ictaluri (13.9 mm to 15.8 mm), A. hydrophila (13.5 mm to 14.1 mm), and P. aeruginosa (13.1 mm to 14.0 mm), compared to a control (+) group with inhibition zones of 25.5 mm to 26.0 mm. Overall, the extract exhibited strong antibacterial activity against these pathogenic bacteria, indicating its potential as a natural antibacterial agent for fish.