Multi-drug-resistant uropathogenic Escherichia coli (UPEC) is considered a significant challenge due to its ability to resist antibiotics and form biofilms. UPEC biofilm formers are well protected and largely inaccessible to antibiotics, which leads to persistent infections and evasion of the host immune system. Understanding how ciprofloxacin and trimethoprim/sulfamethoxazole affect biofilm formation is essential for improving treatment strategies for urinary tract infections (UTIs). A total of 76 UPEC isolates were obtained from Iraqi patients and identified using morphological and biochemical characteristics, as well as the Vitek®-2 Compact system. Minimum inhibitory concentrations (MICs) were determined using the Vitek®-2 system, which is based on CLSI standards, followed by agar diffusion assays to determine MIC, sub-MIC (SMIC), and sub-sub-MIC (SSMIC). A 96-well microtiter plate assay was used to quantify the biofilm-forming ability of UPEC isolates and to evaluate the effects of ciprofloxacin and trimethoprim/sulfamethoxazole on UPEC biofilms. The MICs of ciprofloxacin were ≥ 4 µg/mL for resistant isolates and ≤ 0.25 µg/mL for sensitive ones. For trimethoprim/sulfamethoxazole, MICs were ≥ 320 µg/mL for resistant isolates and ≤ 20 µg/mL for sensitive isolates. Ciprofloxacin inhibited biofilm formation at SSMIC (1 µg/mL) and SMIC (2 µg/mL). Trimethoprim/sulfamethoxazole also showed inhibitory effects, although to a lesser extent than ciprofloxacin. In pre-formed biofilms, ciprofloxacin influenced biofilm integrity at MIC (4 µg/mL), SMIC (2 µg/mL), and SSMIC (1 µg/mL), while trimethoprim/sulfamethoxazole showed variable effects. Both ciprofloxacin and trimethoprim/sulfamethoxazole were capable of inhibiting biofilm formation; however, their efficacy varied. Despite their ability to inhibit initial biofilm formation, ciprofloxacin and trimethoprim/sulfamethoxazole appeared to promote the persistence of already formed UPEC biofilms. Determining the precise concentrations of these antibiotics is essential for effectively managing UTIs caused by