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This study investigated the antibacterial, resistance modulation, biofilm inhibition, and efflux pump inhibition potentials of <i>Loeseneriella africana</i> stem extract and its constituents. The antimicrobial activity was investigated by the high-throughput spot culture growth inhibition (HT-SPOTi) and broth microdilution assays. The resistance modulation activity was investigated using the anti-biofilm formation and efflux pump inhibition assays. Purification of the extract was carried out by chromatographic methods, and the isolated compounds were characterized based on nuclear magnetic resonance, Fourier transform infrared and mass spectrometry spectral data and comparison with published literature. The whole extract, methanol, ethyl acetate, and pet-ether fractions of <i>L. africana</i> all showed antibacterial activity against the test bacteria with MICs ranging from 62.5 to 500.0 µg/mL The whole extract demonstrated resistance modulation effect through strong biofilm inhibition and efflux pump inhibition activities against <i>S. aureus</i> ATCC 25923, <i>E. coli</i> ATCC 25922 and <i>P. aeruginosa</i> ATCC 27853. Chromatographic fractionation of the ethyl acetate fraction resulted in the isolation of a triterpenoid (4S,4αS,6αR,6βS,8αS,12αS,12βR,14αS,14βR)-4,4α,6β,8α,11,11,12β,14α-Octamethyloctadecahydropicene-1,3(2H,4H)-dione) and a phytosterol (β-sitosterol). These compounds showed antibacterial activity against susceptible bacteria at a MIC range of 31–125 µg/mL and potentiated the antibacterial activity of amoxicillin (at ¼ MIC of compounds) against <i>E. coli</i> and <i>P. aeruginosa</i> with modulation factors of 32 and 10, respectively. These compounds also demonstrated good anti-biofilm formation effect at a concentration range of 3–100 µg/mL, and bacterial efflux pump inhibition activity at ½ MIC and ¼ MIC against <i>E. coli</i> and <i>P. aeruginosa</i>. <i>Loeseneriella africana</i> stem bark extracts and constituents elicit considerable antibacterial, resistance modulation, and biofilm and efflux pump inhibition activities. The results justify the indigenous uses of <i>L. africana</i> for managing microbial infections.