Introduction and objective. Infections due to multidrug-resistant (MDR) Enterobacteriaceae are an ongoing global threat in their management. The aim of the study was to investigate the antiimicrobial resistance (AMR) and virulence gene profiles of MDR Gram-negative isolates in Sokoto, northwest Nigeria. Materials and method. A total of 578 clinical samples were collected from patients. Suspected Gram-negative bacteria were isolated from these clinical samples: vaginal swab, pus, stool, blood, wound swab and urine, using Gram-staining and conventional biochemical reactions. These isolates were further identified with an identification kit (Microgen-GN-A), and tested against a panel of 11 antibiotics. A single polymerase chain reaction (PCR) assay targeting 13 virulence gene related to adhesion (fimH, papC, and sfaS), iron chelation (iutA, and fyuA), toxins (astA, stx1, stx2, and eaeA), biofilm (bssS), and serum resistance (traT, iss, and kapsMTII) encoding genes were evaluated. Results. A total of 276 Gram-negative isolates were identified using the Gram stain and biochemical reactions. These organisms were further confirmed with identification kit. Of the 276 isolates, 36 organisms of interest (23 Escherichia coli, 4 Klebsiella pneumoniae and 9 Proteus mirabilis) were identified. Other Gram-negative isolates accounted for the remaining 86.9%. The majority of the isolates were resistant to cefixime (100%) and partially resistant to amikacin (19.4%).The virulence genes bssS (58.3%), fimH (44.4%), and iutA (44.4%) were the most prevalent, whereas kapsMTII (5.6%) and stx2 (2.8%) were least detected, while astA was not detected in any of the isolates. Conclusion. The study elucidated the prevalence of antibiotic resistance and virulence genes in Gram-negative bacteria from clinical isolates in Sokoto, northwestern Nigeria. The majority of the isolates were MDR, thereby posing a public health risk.