Antimicrobial resistance in raw milk is a critical challenge for public health. This study aimed to investigate the in-vitro and in-silico analysis of antimicrobial resistance, mutations, virulence factors (VFs), mobile genetic elements, and diversity of Escherichia coli in cattle raw milk of Gujarat, India during May 2023- April 2024. 56 isolates were recovered from 50 raw milk samples. Among them, 26 were identified as E. coli and subjected to antimicrobial susceptibility testing. The extensively drug-resistant 10 isolates were further confirmed by 16s rRNA identification. Whole-genome Nanopore sequencing was performed on 10 representative strains, and data were further used in bioinformatics analysis. The results revealed that wide array of ARGs and mutations were detected. Plasmid-mediated ARGs were detected in three isolates that include bla