Urinary tract infections (UTIs) are regarded as one of the most serious infections worldwide. Uro Pathogenic E. coli (UPEC) accounts for nearly 80% of UTI infections in females. This study investigated the antibacterial and antibiofilm effects of Lactobacillus acidophilus (l. acidophilus) and Lactobacillus plantarum (lb. plantarum) on multidrug-resistant E. coli obtained from urine samples. Complete bacteriological identification was conducted on 45 E. coli isolated from 80 urine samples of females with UTIs. Antibiotic susceptibility test was performed on all isolates by nine antibiotics. Ten out of the 45 isolates exhibited multidrug resistance (MDR). L. acidophilus and Lb. plantarum showed marked inhibition of MDR E. coli isolates on agar by a diffusion method (16 ± 0.04: 23 ± 0.05 mm). Moreover, L. acidophilus and Lb. plantarum strains inhibited the ability of UPEC to form a biofilm by 56.3% and 39.63%, respectively. The expression of biofilm genes of E. coli are as follows: csgA, crl, csgD showed remarkable downregulation after treatment with probiotics suspension: 0.00364: 0.19078 fold, 0.0005: 0.1894 fold, and 0.0490: 0.0883 for L. acidophilus, respectively. On the other hand, downregulation of biofilm gene expression for csgA, crl, csgD after treatment with Lb. plantarum suspension were expressed by fold changes as follows: 0.0769: 0.3535 fold, 0.05440: 0.12940 fold, and 0.06745: 0.4146, respectively. These findings show that L. acidophilus and Lb. plantarum exhibit potent antibacterial and antibiofilm action against MDR UPEC at both genotypic and phenotypic levels, and appear to be a promising solution in therapeutic applications for recurrent and persistent UTIs.