Escherichia coli O157:H7 has been linked to intestinal infections in humans and is thought to be a major cause of foodborne illnesses. The research is providing an update on the prevalence and toxin profile of multidrug-resistant E. coli O157:H7 in Upper Egypt, from November 2023 to February 2024. A total of 410 samples were obtained from food products, animals, water, and human stool for isolation, identification, and serotyping of multidrug-resistant E. coli O157:H7 by using traditional and molecular techniques. Furthermore, the antibacterial effect of Piper cubeba Linn. (PcL) extract against E. coli O157:H7 was studied in vitro. E. coli and E. coli O157:H7 were found to have a prevalence of 28% and 8%, respectively, with the highest occurrence in human stool. Depending on serology, 12 different E. coli serotypes were identified in the overall samples. The PCR showed that the most virulence genes found were Stx1 (42.4%), followed by uidA (39.4%), eaeA (36.4%), hlyA (30.3%), and Stx2 (15.2%). Furthermore, blaTEM (42.4%), tetA (36.4%), and Aada1 (27.3%) are the most common resistance genes. Most strains were resistant to ampicillin, penicillin G, and tetracycline (100% each), while florfenicol and ciprofloxacin were entirely sensitive. Furthermore, all concentrations of PcL extract (0.5%, 1%, 3%, and 5%) confirmed significant inhibitory activities against E. coli O157:H7 with MIC values of 0.43 mg/mL to 0.74 mg/mL and MBC values of 0.86 mg/mL to 1.76 mg/mL. These results could assist in preserving human health by increasing awareness of the dangers posed by this bacterium and lowering the prevalence of illnesses linked to E. coli O157:H7. This study proposes that PcL extract could be employed as a natural antimicrobial additive in food products, which creates new opportunities for its advancement and application in functional foods.