Urinary tract infection (UTI) is the most common form of extraintestinal pathogenic Escherichia coli (ExPEC) infection. The ability of these ExPEC strains to form biofilms is likely to serve as a pathogenic determinant that allows them to persist in the urinary tract for a prolonged period and contribute to development of UTIs. The widespread use of antibiotics to fight against infectious disease has lead to the emergence of many antibiotic resistant bacteria and the presence of antibiotic resistance genes in bacteria are suggested to affect their ability to form biofilms. In this study the effect of the presence of new antibiotic resistance genes on biofilm formation of UTI associated E. coli strains was examined. Two clinical UTI associated E. coli (SMC18 and SMC 20) were transformed with pGEM-T, pGEM-T::KmΔAmp or pGEM-T::Km to construct ampicillin resistant (Km^SAmp^R), kanamycin resistant (Km^RAmp^S) or ampicillin and kanamycin resistant (Km^RAmp^R) strains. The biofilm forming ability and potential factors associated with biofilm formation (the presence of virulence factors, bacterial surface hydrophobicity, auto-aggregation and bacterial attachment) of these strains were determined. Biofilm formation by E. coli SMC18 was reduced by the presence of either one or both antibiotic resistance gene(s), while biofilm formation of E. coli SMC20 was increased by the presence of the ampicillin resistance gene but not affected by the presence of the kanamycin resistance gene alone or the presence of both ampicillin and kanamycin resistance genes together. The results of the study showed that cells transformed with plasmids carrying different antibiotic resistance genes had significant influence on biofilm formation by different E. coli strains which suggest that the genetic background, in particular the presence of virulence genes, strongly influences biofilm formation by UTI associated E. coli acquiring antibiotic resistance.