Urinary tract infections (UTIs) are the second most prevalent infectious disease with being the most common etiological agent behind these infections, affecting more than 150 million people globally each year. In recent decades, the emergence of multi-drug resistant (MDR) pathogens has rapidly escalated. To combat antimicrobial resistance (AMR), it is important to synthesize new biologically effective alternatives like ionic liquids (ILs) to control the bacterial infection and their spread. Ionic liquids are poorly coordinated organic salts characterized by melting points typically below 100 °C. The ability of ILs to form anionic and cationic interactions contributes to their versatile chemical, physical and biological attributes. In the present study, a total of 9 previously chemically synthesized and characterized ILs were used. For exploration of their antibacterial potential against the urinary tract infections (UTIs) caused by MDR Uropathogenic (UPEC) strains and evaluation of ILs were performed. ILs showed pronounced zone of inhibition (ZOI), minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 29.5 mm, 3.81 μM and 5.08 μM by agar disk diffusion and broth micro-dilution methods, respectively. Scanning electron microscopy results depicted substantial morphological changes in UPEC biofilm formation ascertaining antibiofilm potential of tested ILs. Moreover, ILs showed exceptional antioxidant potential depicted by DPPH assay along with low cytotoxic effect toward mammalian cell lines (NB4), red blood cells and whole blood. Furthermore, the gene expression analysis results justified the antibacterial potential of ILs showing down-regulation of fimH, uvrY and up-regulation of csrA gene in UPEC after ILs treatment. dermal sensitivity assessment also established their non-cytotoxic behavior. analysis validated these results, with the majority of the compounds exhibiting moderate to good absorption.Due to remarkable antibacterial and antioxidant potential and negligible cytoxicity, it could be inferred that ILs could serve as novel antimicrobial alternative agents in the treatment of UTIs.