Catheter-associated urinary tract infections (CAUTIs) are among the most common nosocomial infections globally and represent a significant cause of morbidity and prolonged hospital stays. There is a scarcity of molecular data regarding pathogens and virulence factors linked to CAUTIs, particularly in South Africa. This study aimed to determine the distribution and frequency of uropathogens, including polymicrobial infection patterns, isolated from catheterised patients at the Polokwane National Health Laboratory Services (NHLS), Limpopo Province, from March 2024 to March 2025, and to characterise the prevalence and co-occurrence of six virulence genes (fimH, papC, sfa, hly, cnf, and aer) among Escherichia coli isolates, the predominant uropathogen identified. Specimens from catheterised patients diagnosed with urinary tract infections at the National Health Laboratory Services in Polokwane were used. Uropathogens were isolated on agar and identified using the VITEK^® system. Polymerase chain reaction (PCR) was used for amplification of some virulence genes (fimH, papC, sfa, hly, cnf, and aer) associated with uropathogens. Confirmation of urinary tract infections revealed that Escherichia coli was the predominant uropathogen (32.8%), followed by Klebsiella pneumoniae (17.8%), Enterococcus faecalis, Enterococcus faecium (14%), and others. Among E. coli isolates, virulence gene analysis revealed fimH (79.8%) as the most prevalent, followed by aer (77.4%), sfa (41.7%), hly (27.4%), papC (26.19%), and lastly cnf (9.5%). This study demonstrates that E. coli is the leading cause of CAUTIs in our study population, exhibiting diverse virulence gene combinations that enhance adhesion and overall pathogenicity. The findings emphasise the need for routine molecular surveillance of CAUTI pathogens, reinforcement of catheter-care protocols, and targeted infection prevention strategies.