The global rise in antimicrobial resistance (AMR) has intensified the search for plant-derived alternatives with therapeutic potential. Murraya koenigii (L.) Spreng., a plant valued in traditional medicine, is rich in bioactive compounds, though its bark has not been extensively studied. This research aimed to analyze the phytochemical constituents and evaluate the antibacterial properties of the methanolic extract of M. koenigii bark sourced from the Botanical Garden of VJ’s College of Pharmacy, Rajahmundry. The bark was dried, ground, and extracted using methanol via maceration. Standard qualitative tests were used to identify secondary metabolites such as alkaloids, flavonoids, tannins, saponins, triterpenoids, and cardiac glycosides. The extract’s antibacterial activity was tested against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa using the agar well diffusion method, with Amikacin as the positive control and methanol as the negative control. The extract demonstrated a rich phytochemical profile and showed dose-dependent antibacterial effects. Maximum inhibition was observed at 400 mg/mL with zones of 15.3 mm (S. aureus), 13.8 mm (E. coli), and 12.5 mm (P. aeruginosa). The study indicates that M. koenigii bark methanolic extract possesses significant antibacterial activity, suggesting its potential as a plant-based antimicrobial agent.