Background: The use of plants in the treatment of microbial diseases is increasing worldwide; especially with the increase of bacterial resistance to antibiotics and the development of new diseases that have no cure in modern medicines. Aim: This research was designed to determine the antimicrobial activities of bioactive compounds isolated from Acacia nilotica (A. nilotica) against multi-drug resistant bacteria. Methods: Acacia nilotica samples (i.e., leaves, pods, and back) were collected within Aliero local government area, in Nigeria. Metabolite extraction was performed through maceration. The obtained extract was tested in vitro against multi-drug resistant bacteria that cause diarrhea: Salmonella typhimurium, S. paratyphi, Salmonella sp, Shigella dysenteriae, Shigella sp, and Shigella flexneri, using the agar well diffusion assay. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using the micro-plate serial dilution method. The synergistic (Pod + Leave Steam back) ethanolic crude extract was subjected to column and thin-layer chromatography (TLC) analyses. The obtained fractions were tested against multi-drugs resistance bacteria, and MIC and MBC of the fractions were also determined. Results: The results showed that the synergistic A. nilotica crude extract had the highest mean antibacterial activity recording inhibition zones that ranged from 15.0± 0.58 to 22.7± 0.33 mm. The MIC of ethanolic crude extracts ranged from 100 to 200 mg/ml, while the MBC ranged from 100 to > 400 mg/ ml. Conclusion: The antibacterial potential of the synergistic ethanolic fractions recorded higher activity that ranged from 16.7± 1.20 to 31.0±1.00 mm against all the tested bacteria, compared to the crude extracts.