Objective: To determine the prevalence of selected virulence genes and the antimicrobial susceptibility of multidrug-resistant (MDR) Escherichia coli isolated from diarrheic neonatal calves. Sample: 97 E coli isolates from diarrheic neonatal calves. Procedures: E coli isolates were tested via PCR assay for 6 virulence genes and susceptibility to 17 drugs belonging to 9 classes. A 2-sample test of proportions was used to make comparisons between proportions of virulent and avirulent MDR isolates. Results: 23 of 97 (23.7%) isolates were virulent, and 74 (76.3%) were avirulent. Of the 23 virulent isolates, 15 (65.2%) were positive for K99, 14 (60.9%) for F41, 12 (52.2%) for STa, 9 (39.1%) for Stx1, 6 (26.1%) for intimin, and 0 (0%) for Stx2. Twenty of 23 (87.0%) virulent isolates expressed ≥ 2 virulence genes, and 3 of 23 (13.0%) were positive for 1 virulence factor. Eight of 23 (34.8%) virulent isolates expressed STa, K99, and F41, whereas 1 of 23 (4.4%) was positive for STa, F41, intimin, and Stx1. The second most frequent gene pattern was Stx1 and intimin. Twenty of 23 (87.0%) virulent isolates were MDR; the highest prevalence of resistance was recorded for the macrolide-lincosides, followed by the tetracyclines and penicillins. Also, 17 of 23 (74.0%) virulent isolates were resistant to sulfadimethoxine, and 10 of 23 (43.5%) were resistant to trimethoprim-sulfamethoxazole. Additionally, 60 of 74 (81.0%) avirulent isolates were MDR. Conclusions and Clinical Relevance: The prevalence of multidrug resistance was comparable for virulent and avirulent E coli isolated from diarrheic neonatal calves. Cephalosporins and aminoglycosides had reasonable susceptibility.