Several types of bacteria enhance their survival by attaching to non-living surfaces or tissues, presenting them as multicellular communities covered by a protective extracellular matrix called biofilm. There has been a clear interest in assessing the relationship between antibiotic resistance phenotype and biofilm production. The aim of this paper was to present additional experimental results on this topic and to test the biofilm formation ability of Escherichia coli isolates using in vitro methods in the context of their antibiotic resistance. In our case study, (24) isolates of E. coli were included, and the colony morphology of the isolates was recorded after 24 hours, while the antimicrobial susceptibility test was performed by adhesion using the Kirby Power disc diffusion method. The biofilm formation of the isolates was evaluated using the method. All 24 clinical specimens 100% showed strong biofilm formation and all clinical specimens showed the presence of pml gene by clear bands in gel electrophoresis. The results of the antibiotic sensitivity test of E. coli isolates by disk diffusion method against 18 types of commonly used antibiotics showed a large variation in their rates of antibiotic resistance, as the highest percentage of resistance to the antibiotic to E. coli resistance to the most common antibiotics used in treatment. The highest rate of resistance was seen with cephalothin and Clindamycin 24/24 (100%) followed by ceftazidime, cefotaxime Levofloxacin, Ciprofloxacin and Erythromycin 18/24 (75%), Netilmicin, Chloramphenicol, Amoxicillin, and Tetracycline 16/24 (66%), Gentamicin 14/24 (58%), Tobramycin and Trimethoprim 11/24 (45%), Ampicillin and Impenem 6/24 (25%), Colistin sulfate and Azithromycin 4/24 (16%) as all isolates were sensitive. The study found a positive correlation between interesting study genes and biofilm formation and antibiotics in E. coli. Strains that appeared multidrug-resistant (MDR) were given a high mean of biofilm. It has been demonstrated that some correlations exist between antibiotic resistance and the biofilm-forming ability of E. coli isolates. There is an association between biofilm production with persistent infection and antibiotic failure.