The majority of common Escherichia coli species is related to humans and cause extra-intestinal infections. There is a link between majority of these infections and bacterial CTX-M. The present study was conducted to investigate the relationship between biofilm formation and CTX-M gene expression in clinical E. coli isolates. Thirty-five clinical isolates of E. coli were obtained from different clinical samples including urine, blood, wounds, and burns from four hospitals in Baghdad. Antibiotic susceptibility and biofilm formation tests were carried out. Polymerase chain reaction (PCR) and reverse transcriptase-PCR were used to detect and evaluate the expression of the CTX-M gene. The isolates were resistant to cephalosporins, aminoglycosides, and carbapenems with values of 94.3, 60, and 14.3%, respectively. There was a significant (p <0.01) relationship between biofilm formation and resistance to multiple antibiotics in the E. coli isolates. The PCR assay revealed that 94.3% of the isolates have the CTX-M gene, but no significant correlation between the biofilm values and the concentrations of the CTX-M gene expression. The findings of this study suggest that these antibiotics can be used in the empirical treatment of E. coli-caused infection-linked biofilm formation, and the antibiotic sensitivity pattern aids in antibiotic selection.