The ability of pathogenic Escherichia coli to produce carbapenemase enzymes is a characteristic that allows them to resist various antibiotics, including last-resort antibiotics like colistin and carbapenem. Our objectives were to identify rapidly developing antibiotic resistance (AR), assess β-lactamases production, and detect mcr-1 and blaNDM-1 genes in the isolates. A prospective cross-sectional study was carried out in a referral hospital located in Kathmandu from November 2019 to December 2020 using standard laboratory and molecular protocols. Among 77 total E. coli isolates, 64 (83.1%) of them were categorized as MDR. Phenotypically 13 (20.3%) colistin-resistant, 30 (46.9%) ESBL and 8 (12.5%) AmpC producers, and 5 (7.8%) ESBL/AmpC co-producers were distributed among MDR- E. coli. Minimum inhibitory concentrations (MIC) against the majority of MDR isolates were exhibited at 1 g/L. Of these 77 E. coli isolates, 24 (31.2%) were carbapenem-resistant. Among these carbapenem-resistant bacteria, 11 (45.9%) isolates were reported to be colistin-resistant, while 15 (62.5%) and 2 (8.3%) were MBL and KPC producers, respectively. Out of 15 MBL producers, 6 (40%) harbored blaNDM-1, and 8 (61.5%) out of 13 colistin-resistant pathogens possessed mcr-1. The resistance by colistin- and carbapenem were statistically associated ( P < .001). However, only 2 (18.2%) of the co-resistant bacteria were found to have both genes. Our study revealed the highly prevalent MDR and the carbapenem-resistant E. coli and emphasized that the pathogens possess a wide range of capabilities to synthesize β-lactamases. These findings could assist to expand the understanding of AR in terms of enzyme production.