The modern era has used new phytochemical compounds to face the problem of antimicrobial resistance all over the world. The present study aimed to evaluate the antimicrobial and antibiofilm activity of Peltophorum africanum leaf methanolic extract against pathogenic isolates. To achieve this, clinical isolates of Acinetobacter baumannii, Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus were obtained from Al-Demerdash Hospital where their antimicrobial resistance profiles were assessed according to CLSI guidelines using Kirby Bauer disc diffusion method. The calculated minimal inhibitory concentrations (MIC) of the crude extract against the collected isolates ranged between 23 ± 1.1 and 7.8 ± 0.9 with the highest observed activity against MRSA isolates. MIC was determined by the agar well diffusion. Antibiofilm activity was evaluated using the crystal violet method against three strong biofilm producers and showed that the extract exhibited no antibiofilm activity. The paper also investigated its anticancer potential against the human colon cancer cell (Caco-2) line. In conclusion, it can be inferred that; Peltophorum africanum leaf methanolic extract has a good potential to be used in treating infections caused by methicillin-resistant Staphylococcus aureus with probably a high safety profile.