Infectious diseases caused by bacteria constitute the main cause of morbidity and mortality throughout the world and mainly in developing countries. In this work, the influence of fractioning and the mode of action of stem barks methanol extract of Enantia chlorantha were investigated. The aim was to optimize the antibacterial activity of the methanol extract. The extract was prepared by maceration of barks powder in methanol. Fractioning was done using increasing solvents polarity. Standard phytochemical methods were used for phytochemical screening. Minimum Inhibitory Concentrations (MIC) and Minimum Bactericidal Concentration (MBC) of the methanol extract and fractions were determined using broth microdilution method. The studied mode of action of both methanol extract and n-butanol fraction included antibiofilm activity, H+-ATPase-mediated proton pumping assay, salt tolerance, and cells cycle. The methanol extract of E. chlorantha stem barks was found to be active on all the bacteria tested (32 ≤ MIC ≤ 512 μg/mL), its activity being significant (MIC < 100 μg/ml) out of 5 of the 28 clinical isolates used. Salmonella enterica serovar paratyphi A was the most sensitive (32 μg/mL). Compared to the extract and other fractions, the n-butanol fraction was found to be more active (32 ≤ MIC ≤ 256). Significant antibacterial activity of this fraction was observed out of 10 of the 28 bacterial isolates and 3 out of 7 bacterial strains. Lowest MIC values (32 μg/ml) of this fraction were obtained with Escherichia coli (136), Pseudomonas aeruginosa (CIP 76110), and Salmonella enterica serovar typhi 9. The methanol extract of E. chlorantha and its n-butanol fraction revealed several modes of action including the prolongation of the latency phase of the bacterial growth, the inhibition of the pump with protons H+ - ATPases bacterial, the loss of the salt tolerance of the Staphylococcus aureus, and inhibition of the formation of the bacterial biofilm. The present results showed that the n-butanol fraction of the methanol stem barks extract of E. chlorantha possess the essential antibacterial components and could best be used to fight against bacterial infections as compared to methanol extract.