Urine samples collected from hospitalized male and female patients belonging to different age groups suffering from urinary tract infections were used in the present study. The samples were subjected to isolate the strains of E.coli. The isolates were found to be capable of producing biofilm and Extended Spectrum β –Lactamases (ESBLs) especially of CTX – M - type. Both male and female affected individuals exhibited high incidence of E.coli in the urine samples. The overall results indicated that the biofilm producing E.coli strains with β – lactamase activity had highest antibiotic resistance.