Background: Uropathogenic E. coli (UPEC) are important clinical pathogens and are considered as one of the major causes of urinary tract infection. Unfortunately, the treatment of UPEC infections is becoming more difficult because isolates are increasingly resistant to commonly used antimicrobial agents. The potential ability of UPEC to form biofilm might explain its outstanding antibiotic resistance and survival properties. Objectives: We aimed to assess the effect of cell free supernatants (CFS) of Lactobacillus acidophilus La-5 and Lactobacillus helveticus B-734 on the ability of ESBL-producing UPEC isolates to form biofilm in vitro and to eradicate the already formed biofilm of UPEC isolates. Methodology: A total of 50 isolates of ESBL-producing UPEC, were recovered from different private laboratories, then subcultured on MacConkey's medium and identified by conventional microbiological methods. Antibiotic susceptibility testing was performed by Kirby-Bauer disc diffusion method and confirmed for ESBL production. The ability of UPEC to produce biofilm was determined using Congo red agar (CRA) method and tissue culture plate (TCP) method. CFS of L. acidophilus La-5 and L. helveticus B-734 were prepared and used to assess its effect on the ability of ESBL-producing UPEC isolates to form biofilm in vitro and the eradication of the already formed biofilm. Results: Biofilm formation was found in 28% of the isolates. Furthermore, the strong biofilm-forming category was detected in 10% of the isolates, whereas the moderate biofilm-forming category was detected in 18%. CFS of L. acidophilus La-5 was more effective (91.12%) in inhibiting the formation of E. coli biofilm than CFS of L. helveticus B-734 (76.44%), while it was almost the same efficacy (83%) regarding eradication of E. coli biofilm. All ESBL- producing UPEC isolates were sensitive to cefoxitin (100%) followed by nitrofurantoin (98%) and imipenem (94%). Conclusion: L. acidophilus La-5 and L. helveticus B-734 may be a new therapeutic options for UTI caused by UPEC.