Abstract: Medicinal plants serve as sources of valuable compounds with therapeutic potential. Plumeria rubra L. (Apocynaceae) is a medicinally important tree which has been reported as ethnomedicinal cure of different ailments. In the current investigation, n-hexane fraction of crude methanolic extract of P. rubra stem bark was investigated against four ATCC (American type culture collection) bacterial strains Staphylococcus aureus (ATCC 25923), Enterobacter cloacae (ATCC 13047), Pseudomonas aeruginosa (ATCC 27853) and Serratia marcescens (ATCC 13880). Inhibitions of the plant extract against all the four microorganisms were tested by both agar-diffusion assay and broth microdilution method. The n-hexane fraction of crude methanolic extract of P. rubra stem bark showed MICs of 13.5, 11.8, 8.5 and 16.9 mg mL-1 and induced a maximum of 91.53, 92.84, 94.69 and 85.29% growth inhibition against S. aureus (ATCC 25923), E. cloacae (ATCC 13047), P. aeruginosa (ATCC 27853) and S. marcescens (ATCC 13880), respectively. The active plant extract in this study showed significant antibacterial activities against all the human pathogenic strains, adding credence to the ethnomedicinal uses of the plant, as well as, suggesting towards its specific use against the tested microorganisms.