The presence of Escherichia coli, Staphylococcus aureus, Listeria monocytogenes and Salmonella spp. was determined in 75 samples of conventional beef and in 75 samples of organic beef. All samples came from cattle slaughtered and processed in the same slaughterhouse and quartering room. A total of 180 E. coli, 180 S. aureus and 98 L. monocytogenes strains were analyzed by an agar disk diffusion assay for their resistance to 11 antimicrobials, for the case of E. coli and S. aureus, or 9 antimicrobials, for the case of L. monocytogenes. Salmonella spp. were not isolated from any of the beef samples. No significant differences in prevalence were obtained for any of the bacterial species tested between organic and conventional beef. E. coli isolated from organic beef exhibited significant differences in antimicrobial resistance against 5 of the 11 antimicrobials tested as compared to isolates recovered from conventional beef. In the case of S. aureus, these differences were only found for 3 of the 11 antimicrobials tested and for L. monocytogenes, no differences were obtained between isolates obtained from organic or conventional beef. Although no significant differences were obtained in microbiological contamination, E. coli and S. aureus isolates from organically farmed beef samples showed significantly lower rates of antimicrobial resistance in E. coli and S. aureus isolates.