Urinary tract infections (UTIs) are among the most common clinical diseases worldwide, affecting around 150 million people annually. Despite extensive efforts, the prevalence of UTIs remains high. This study aimed to investigate antibiotic resistance and virulence genes in pathogenic Escherichia coli (E. coli) isolated from UTI patients at Al-Hussein Hospital, Nasiriyah City, Iraq. A total of 250 urine samples were collected, with 77% of patients being female and 23% male. Age distribution included 13% under 20 years, 33% aged 20-40, 35% aged 40-60, and 19% aged 60 years or older. The most common UTI types included 20% complicated UTIs, 25% uncomplicated UTIs, 30% community-acquired UTIs, and 25% healthcare-associated UTIs. Among the E. coli isolates, antibiotic resistance was observed, with ceftizoxime (CZX 30 mcg) showing the lowest resistance at 21.6%, followed by ceftriaxone (CTR 30 mcg) at 26%, cefpirome (CFP 30 mcg) at 22.8%, and cefepime (CPM 30 mcg) at 12.4%. The highest resistance was found with cefadroxil (CFR 30 mcg) at 30.8%. Additionally, the study detected several virulence and resistance genes, including papAH (8%), saf (10.8%), kps (8%), yfcV (12%), ST131 (20.4%), VAT (11.6%), OqxA (6%), and bla_CTX-M (7.2%). These findings emphasize the need for better understanding the genetic makeup of E. coli in UTIs, aiding in the development of diagnostic and treatment strategies.