Colibacillosis is an infectious disease in animals, which is caused by Escherichia coli, leading to economic losses. The current investigation was conducted to evaluate the molecular typing of colibacillosis-associated Escherichia coli for determination of O157:H7 serotype and virulence factors. Overall, 100 faecal samples were screened between November 2023 to February 2024 from in and around Jaipur district of Rajasthan. Sample screening was done for isolating E. coli in which 77 isolates were recovered from 100 diarrhoeic cattle and its serological identification indicated presence of the most frequent serotype, encompassing O76 (5), O3 (4), O7 (4), O16 (4), O50 (4), O111 (3), O113 (2), O125 (2), O135 (2), O2 (1), O4 (1), O9 (1), O19 (1), O27 (1), O66 (1), O85 (1), O109 (1), O114 (1) and O156 (1). Finally, Molecular characterization of the recovered isolates was conducted using PCR for detecting presence of fliCH7, rfbO157, stx1, stx2, EhlyA along with eaeA genes associated with E. coli. Among 77 E. coli isolates, none were recognized as O157:H7 serotypes. Among total E. coli isolates, 40 comprised Shiga toxin-producing E. coli (STEC) genes. The stx2 gene was the most prevalent (32.5%), followed by stx1 (17.5%), while combinations involving stx1, stx2 and EhlyA were detected in 10%-12.5% of isolates. The eaeA gene was rare, occurring in only 2.5% of the virulence-positive isolates. Results suggest that conventional culture methods, along with serological and molecular assays, are key techniques for isolating and characterizing STEC from fecal samples of diarrheic cattle.