Background <p>Nanoparticles (NPs) have attracted increasing attention because of their antimicrobial properties. This study aimed to evaluate the antibacterial and antibiofilm activities of nickel oxide (NiO) nanoparticles against representative uropathogenic <i>Escherichia coli</i> (UPEC) O-serotypes isolated from patients with urinary tract infections (UTIs) and characterized by ERIC-PCR typing.</p> Methods <p>A total of 153 UPEC isolates were characterized by PCR-based O-serogrouping and ERIC-PCR typing. NiO nanoparticles were synthesized using the sol-gel method. The antibacterial and antibiofilm activities of NiO nanoparticles were evaluated against one representative biofilm-forming isolate from each ERIC type using the broth microdilution and microtiter plate methods, respectively.</p> Results <p>Among the 153 UPEC isolates, seven O-serogroups, O2, O4, O6, O8, O15, O16, and O25, were detected, with O25 being the most prevalent. ERIC-PCR analysis classified the isolates into 19 distinct clusters (E1–E19) at an 80% similarity threshold, comprising 13 common types (each containing ≥ 2 isolates) and 6 unique (singleton) types. No significant association was observed between O-serogroups and ERIC-PCR clusters (<i>p</i> &gt; 0.05). NiO nanoparticles exhibited antibacterial and antibiofilm activity against 19 representative UPEC isolates, with MIC values ranging from 250 to 500&#xa0;µg/mL and significant inhibition of biofilm formation at concentrations of 125–500&#xa0;µg/mL (<i>p</i> = 0.001). Biofilm inhibition was also observed at sub-MIC concentrations.</p> Conclusions <p>NiO nanoparticles demonstrated in vitro antibacterial and antibiofilm activity against 19 representative UPEC isolates. However, broader isolate screening and comprehensive toxicity and in vivo studies are required to further evaluate the antimicrobial activity and potential applicability of NiO nanoparticles.</p>