Background: The beneficial gut bacterium E. coli can cause blood poisoning, diarrhoea, and other gastrointestinal and systemic disorders. Objective: This study amid to examines the antibiofilm activity of Laurus nobilis leaves extract on E. coli isolates and compares pre- and post-treatment gene expression of fimA and papC genes. Subjects and Methods: Ten isolates of E. coli were obtained from the Genetic Engineering and Biotechnology Institute, University of Baghdad, which was previously collected from Baghdad city hospitals and diagnosed by chemical tests, the diagnosis was confirmed using VITEK-2 System. The preparation of the aqueous and methanolic Laurus nobilis leaves extracts was done by using the maceration method and Soxhlet apparatus respectively. HPLC were conducted to determine the active compounds in the extracts. Moreover, molecular detection of fimA and papC genes and analysis of the gene expression by comparing the isolates treated with sub MIC of methanolic L. nobilis leaves extract with the untreated isolates. Results: Methanolic and aqueous extracts contained alkaloids, tannins, phenols, saponins, flavonoids, and glycosides. Seven polyphenolic compounds, four flavonoids derivatives (Apigenin, Luteolin, Rutin, and kaempferol) and three phenolic acids (Caffeic acid, Gallic acid, and Syringic acid), were identified by matching retention time with the standards. Laurus nobilis methanolic leaf extract inhibited 90% and 100% of E. coli biofilm development at 32 and 64 mg/ml. Conclusion: The result of the gene expression revealed that there is a decrease in the expression of the fimA and papC genes. The present study concluded that the Laurus nobilis leaves extract have rich phytochemical contents, so the methanolic extract had an excellent reduction effect on biofilm formation and showed remarkable down-regulation on the papC and fimA genes, which are responsible for the biofilm formation in E. coli.