Characterization of antimicrobial resistance and virulence gene profiles provides important information on the potential pathogenicity of bacteria. This information can be used to facilitate prompt and effective treatment of bacterial infections. We developed and tested a PCR-based microarray platform for detecting virulence and antimicrobial resistance genes in Salmonella serovars and Escherichia coli. Twelve Salmonella and seven E. coli isolates were screened for the presence of 25 virulence and 23 antimicrobial resistance genes. All S. Typhimurium DT104 isolates harbored virulence plasmids. E. coli O157:H7 isolates possessed virulence genes typical of enterohemorrhagic E. coli (EHEC), whereas E. coli O126 isolates contained virulence genes characteristic of enteropathogenic E. coli (EPEC) and E. coli O111, O78 and O147 isolates had virulence genes characteristic of enterotoxigenic E. coli (ETEC). Correlation between antimicrobial resistance phenotype and genotype was observed for each isolate. The aadA, tetA, and sulI genes were most commonly detected in bacteria resistant to streptomycin, tetracycline and sulfonamide, respectively. All isolates exhibiting resistance to third generation cephalosporins harbored the bla(CMY-2) and bla(TEM-1) genes. Microarray analysis is an effective method to rapidly screen Salmonella and E. coli for multiple antimicrobial resistance and virulence genes.