We assessed the in vitro antimicrobial activity of Peltophorum africanum by means of the agar well and macrodilution methods. The toxicity on a normal human liver cell (Chang liver cell) was determined using the CellTiter-Blue cell viability assay, and the compounds contained in the fractions were identified using GC-MS. Zone diameter of inhibition of the extract ranged from 12.5 ± 0.7 to 32 ± 2.8 mm for bacteria and from 7.5 ± 0.7 to 26.4 ± 3.4 mm for yeast. Marked activity of the extract was observed against Plesiomonas shigelloides ATCC 51903, with MIC and MLC values of 0.15625 and 0.3125 mg/mL, respectively. The extract was both bactericidal (MIC(index) ≤ 2) and bacteriostatic/fungistatic (MIC(index) > 2) in activity. Lethal dose at 50 (LD50) showed 82.64 ± 1.40 degree of toxicity at 24 hrs, and 95 percentile of cell death dose activity ranged from log 3.12 ± 0.01 to 4.59 ± 0.03. The activity of the eight fractions tested ranged from 1.0 ± 0.5 to 3.7 ± 1.6 mg/mL (IC50) and from 2.1 ± 0.8 to 6.25 ± 0 mg/mL (IC90). The extract was toxic to human Chang liver cell lines.