This study investigated the prevalence and characterization of Enterococcus species isolated from food sources in Japan and Egypt using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). A total of 198 food and environmental samples were analyzed, yielding 67 Enterococcus isolates. MALDI-TOF MS accurately identified 98.5% of the isolates and was further used to classify strains based on protein fingerprinting. The study revealed a high prevalence of the virulence genes efaA and gelE, while the cylA gene was absent in all isolates. Among E. faecalis isolates, 84.75% were resistant to kanamycin and 78% exhibited multidrug resistance, whereas moderate vancomycin resistance was detected only in Egyptian isolates. Biofilm formation was positively associated with the presence of multiple virulence genes. Overall, the study demonstrated that MALDI-TOF MS is a rapid and reliable tool for identifying and characterizing foodborne Enterococcus strains and provides valuable information on their pathogenic potential and antimicrobial resistance.