Objective.The present study was carried out to establish the chemical profile of the methanolic extract ofPolyalthia longifoliastem bark and investigate its antibacterial property against some human pathogenic bacteria.Methods.The extract was analysed using liquid and gas chromatography coupled to mass spectrometry. Antibacterial activity ofP. longifoliaextract against some human pathogenic bacteria was screened using the AlamarBlue method, and MIC and MBC were determined.Results and Conclusion.Liquid chromatography-mass spectrometry (LC-MS) revealed the presence of 21 compounds among which 12 were identified. Gas chromatography-mass spectrometry (GC-MS) allowed identification of 26 compounds, the three major ones being the following: cis vaccenic acid (17.79 %), 3-ethyl-3-hydroxyandrostan-17-one (13.80 %) and copaiferic acid B (12.82 %).P. longifoliaextract was active against Gram-positive bacteria with MIC ranging from 1 to 2 mg ml−1and MBC from 2 to 6 mg ml−1. This study demonstrated the bactericidal effect of the methanolic extract ofPolyalthia longifoliastem bark against some human pathogenic bacteria, including methicillin-resistantS. aureus. This effect could be related to the presence in the extract of a broad diversity of well-known compounds with established pharmacological properties. These results support the ethnomedicinal use ofP. longifoliastem bark in Cameroon for the management of methicillin-resistantS. aureus(MRSA)-related infections.