This study investigated the prevalence, genetic diversity, virulence genes, and antimicrobial resistance of Bacillus cereus sensu stricto isolated from various food sources in Japan. Among 179 food and environmental samples, 22.34% tested positive for B. cereus. Molecular characterization revealed 34 sequence types, including 23 novel sequence types, indicating high genetic diversity. All isolates carried at least one virulence gene, whereas the cytK gene was absent from all strains. Antimicrobial susceptibility testing demonstrated that all isolates were multidrug-resistant, exhibiting particularly high resistance to β-lactam antibiotics and vancomycin while remaining fully susceptible to gentamicin. The isolates also differed in their biofilm-forming abilities. These findings highlight the public health risks associated with multidrug-resistant B. cereus in food products and emphasize the need for continuous surveillance and effective food safety control measures.