Shiga toxin-producing E. coli (STEC) are considered important zoonotic pathogens of great economic significance, associated with diarrhea, hemolytic uremic syndrome (HUS), hemorrhagic colitis (HC), and death in humans. This study aimed to investigate the distribution of various STEC virulence gene markers and antimicrobial susceptibility (AST) profiles associated within E. coli isolates from the recto-anal mucosal swabs (RAMSs) of slaughtered cattle and buffaloes in Islamabad, Pakistan. The RAMSs (n = 200) were analyzed using multiplex PCR for the presence of stx1, stx2, eae, and ehxA genes. Samples that were positive for one or more of the virulence genes were inoculated with Sorbitol MacConkey agar (SMAC) for isolation of STEC. The isolates were further analyzed for the presence of virulence genes using multiplex PCR. Of the 200 RAMS, 118 (59%) were positive for one or more virulence genes. E. coli isolates (n = 18) with one or more virulence genes were recovered from the 118 positive samples. The DNA of the isolates positive for one or more virulent genes was extracted and subjected to whole genome sequencing using Illumina. Analysis of the WGS data indicated that the E. coli isolates could be differentiated into 11 serotypes. Most E. coli isolates (13/18; 72.2%) carried five genes (stx1, stx2, Iha, iss, and IpfA) in various combinations. In addition to these five genes, other virulence genes identified in these isolates were espI, ireA, espP, exhA, epeA, mcmA, mch, ast, celB, eilA, katP, and capU. The AST was performed using the Kirby–Bauer disk diffusion test. The study indicated that all the isolates were resistant to rifampicin and a significant proportion of the isolates were MDR. A wide range of antimicrobial resistance genes (ARGs) were detected among the isolates, reflecting the complex nature of resistance mechanisms. The study results indicate that cattle and buffaloes slaughtered in Islamabad might be the carriers of antimicrobial resistant STEC of zoonotic significance, thus representing a source of human infection.