A total of 27 samples from 9 different ghat locations along the course of Krishna River (in triplicate) in the Vijayawada city landscape were collected and analysed for presumptive coliform tests. From the positive presumptive coliform test samples, 44 E. coli isolates were recovered and confirmed by biochemical tests and molecular tests targeting 16s rRNA. All 44 E. coli isolates were further subjected for the detection of virulence genes i.e. stx_1, stx_2, eaeA and hlyA genes using multiplex PCR. Out of 44 isolates two isolates carried all four genes, one isolate harbored stx_2, eaeA and hlyA genes and one isolate showed the presence of eae and hlyA genes in combination. The 44 E. coli isolates were subjected to antimicrobial sensitivity/resistance against 12 antimicrobial agents by disc diffusion method. Higher resistance was observed for naladixic acid (40.9%) followed by ampicillin (38.63%), ceftazidime (34%) and ceftriaxone (27.27%). Out of 44 isolates of E. coli, ten isolates harbored β-lactamase genes, with bla _TEM being predominant gene detected (15.9%, 7/44). ERIC and REP-PCR analysis revealed a greater degree of genetic diversity among the ten β-lactamase producing E. coli isolated from Krishna River water.