Background: Escherichia coli is a Gram-negative, opportunistic human pathogen in which increasing antibiotic resistance is a great concern for continued human survival. Although biofilm formation is a mechanism that helps E. coli to survive in unfavorable conditions, according to the importance of biofilm formation in developing the antibiotic resistance here, we studied the relation between antibiotic resistance and in vitro qualitative rating method biofilm formation in E. coli isolated from patients with urinary tract infection (UTI). Materials and Methods: The clinical isolates of E. coli (n = 100) were collected from urine of patients with UTI attending Isfahan Alzahra hospital. The strains were confirmed as E. coli using biochemical tests and molecular method. The Kirby-Bauer disk diffusion tests were done according to the Clinical and Laboratory Standards Institute protocol, and the biofilm synthesis was performed by microplate method. The binary logistic test was applied and P < 0.05 was considered statistically significant. Results: Our results showed a high outbreak of multidrug-resistant (MDR) E. coli strains (73%) and the highest resistance was observed toward ampicillin. The prevalence of biofilm producer isolates was 80% that 29% produced strong biofilm. The distribution of non-MDR isolates was high among strong biofilm producers, which shows a significant negative correlation between biofilm production and MDR pattern (P < 0.001). Conclusions: We found a negative correlation between MDR phenotype and biofilm formation capacity. This transmits the concept that more antibiotic susceptibility of strong biofilm producers may be due to the reduced exposure to multiple antibiotics.