This present study investigates the in vitro inhibitory effect and antimicrobial activity of secondary metabolites isolated from the roots of Khaya senegalensis, a plant of the Meliaceae family. Khaya senegalensis is widely used in traditional medicine for the treatment of various illnesses such as: fever, stomach ache, diarrhea, dysentery and anemia. The chemical study of the extract with CH 2 Cl 2-MeOH (1:1) led to the isolation of five compounds: Alphitolic acid (1); Epigouanic acid (2); Methyl angolensate (3); Rohituca-3 (4) and 5, 6, 7, 3′, 4′-pentamethoxyflavone or Sinensetin (5). Alphitolic acid (1), Rohituca-3 (4) and 5, 6, 7, 3′, 4′-pentamethoxyflavone or Sinensetin (5) were isolated from the roots of this plant for the first time. The structures of the isolated compounds have been elucidated on the basis of spectroscopic analysis and a comparison of their spectral data with those reported in the literature. The results of the antibiogram tests showed that the strain of Escherichia coli is sensitive to all the antibiotics tested except Ceftazidime, a Cephalosporin. The Staphylococcus aureus strain is resistant to almost all the antibiotics tested except Amikacin, an aminoglycoside. This is because the enzymes diffuse through the inter and intraspecific transmission of genes through a plasmid. The antibiogram made it possible to establish the sensitivity profile of the strains tested with regard to certain antibiotics. The antimicrobial tests carried out showed that the inhibitory effect of the compounds isolated from Khaya senegalensis on the four bacterial strains tested at the concentration of 25 mg/mL positively influenced at least one of the microbial strains. However, compounds 1, 2 and 3 did not show any bacterial growth inhibitory activity against Proteus vulgaris. MIC obtained for microbiological tests varied between 0.097 and 0.195 mg/mL for the most sensitive strains of Escherichia coli and Pseudomonas aeruginosa, which revealed the highest antibacterial powers. Furthermore, these results therefore show a great variability in the bacteriostatic qualities of the compounds with respect to the different strains. The two Grampositive strains of Staphylococcus aureus are more sensitive than the other Gram-negative bacterial strains tested. From the antibacterial activity, it appears that the compounds isolated from this plant have a bactericidal activity against Escherichia coli and Pseudomonas aeruginosa. This bactericide could justify their use in herbal medicine against bacterial infections.