Background and objectives Uropathogenic E.coli (UPEC) are major causative agents of urinary tract infection (UTIs), they often possess strong biofilm-forming abilities, and capable of resisting many antibiotics, making catheter associated UTIs (CAUTIs) difficult to treat. Essential oils such as tea tree oil (TTO) have emerged as natural alternatives to antibiotics. This study aimed to evaluate the antibacterial, adhesion, and biofilm-forming efficacy of TTO against UPEC, while analyzing its effect on the gene expression of csgA gene and determining its cytotoxicity. Methods Four UPEC isolates collected from different UTIs patients from Baghdad Province. The Antibacterial activity of TTO evaluated using agar wells diffusion assay and micro dilution using resazurin. Anti-adhesion and anti-biofilm were assessed using silicon Foley catheters. The csgA encode to curli fibers determined using polymerase chain reaction (PCR) and gene expression measured using qPCR. Cytotoxicity of TTO measured against renal carcinoma (A498) and normal fibroblast (HdFn) cell lines via MTT assay. Results TTO inhibited UPEC with inhibition zone diameter of 12–25 mm (p < 0.0001) and MIC value was 0.25%. In Foley catheter model, the concentrated TTO reduced adhesion and biofilm formation (p < 0.0001). csgA harbored within all subjected isolates. Real time quantitative PCR (RT-qPCR) revealed significant (p < 0.0001) upregulation within susceptible isolates (2.2) fold change. Cytotoxicity via MTT assay reveled selective activity of TTO on (A498, IC50= 265.8 µg/mL) over (HdFn, IC50 = 852.5 µg/mL; p < 0.0001). Conclusion TTO demonstrated potential antibacterial, anti-adhesion and anti-biofilm activity against UPEC along with modulations of csgA gene expression and selective cytotoxicity