Background: The emergence of multiple antibiotic resistance (MAR) Escherichia coli (E. coli) and virulent non-O157 Shiga toxin-producing Escherichia coli (STEC) poses a growing concern to the meat industry. Non-O157 STEC strains including O26, O45, O103, O111, O121, and O145 have been implicated in the occurrence of bloody diarrhea and hemolytic uremic syndrome in humans. This research assessed prevalence, matrix-assisted laser desorption/ionization-time of flight mass-spectrometry (MALDI-TOF MS) protein mass-spectra profiles, multidrug-resistance traits, polymerase chain reaction detection of virulence, and antibiotic-resistance genes of E. coli isolated from beef carcasses and slaughterhouse environments. Methods: A total of 180 convenience sponge samples were collected from two different sources-specific parts of beef carcasses and surfaces of the processing environment at the slaughterhouse of Ha′il, Saudi Arabia between September and November 2020. MALDI BioTyper and phylotype-based identification methods accurately identified and classified the samples as belonging to the genus belonging to the Escherichia coli domain of bacteria (NCBI txid: 562). Results: Expected changes were seen in the mass peak spectrum defining nine closely related isolates and four unrelated E. coli isolates. Serological typing of E. coli revealed enterotoxigenic E. coli O166 (19.10%); enteropathogenic E. coli O146 (16.36%) and O44 (18.18%); enterohemorrhagic E. coli O111 (31.18%) and O26 (14.54%). Forty-five percent of examined E. coli were resistant to seven antimicrobials; 75% of 20 selected isolates were resistant to three or more antimicrobials. phoA and blaTEM genes were detected in all selected E. coli isolates. Conclusion: This study confirmed the efficiency and validity of Matrix-assisted laser desorption/ionization time of flight mass-spectrometry in screening for multi-drug resistant E. coli isolated from slaughterhouse derived beef carcasses in Ha’il, Saudi Arabia. We contributed by revealing the distinction between related and non-related strains of E.coli in livestock. The findings in this study can inform improved policy development decision making and resource allocation related to livestock processing regarding antimicrobial use in food animals and rapid screening for effective multiple antibiotic resistance E. coli and virulent non-O157 STEC control in the slaughterhouses