Multidrug resistance is one of the worldwide public health concerns. Water represents the most suitable environment, for the exchange of antibiotic resistance genes among pathogenic to non-pathogenic bacteria. Therefore, we aimed to screen the presence of bla_NDM-1, bla_TEM, bla_SHV, bla_CTX-M and mcr1–5 genes among water samples from different locations of Lower Himachal Pradesh. We examined the genotypic incidences of bla_NDM-1, bla_TEM, bla_SHV, bla_CTXM and mcr1–5 by polymerase chain reaction. Survivability assay, fitness cost assay and biofilm assay were performed for phenotypic characterization. The presence of bla_NDM-1 and its related variants were analysed and confirmed by sequencing-based approaches. A total of 73 bacterial strains were identified on M-lauryl sulphate agar medium. Out of 73 colistin-resistant isolates, 34 were E. coli and 39 were K. pneumoniae. Out of 34 samples, 2 (5.8%), 2 (5.8%), 5 (14.7%), 5 (14.7%) and 4 (11.76%) E. coli were bla_TEM, bla_SHV, bla_CTXM-1, bla_CTXM-2 and bla_CTXM-15 positive, respectively. Among 39 K. pneumoniae, 15 (38.4%), 6 (15.3%), 10 (25.6%), 9 (23.07%) and 10 (25.6%) were bla_TEM, bla_SHV, bla_CTXM-1, bla_CTXM-2 and bla_CTXM-15 positive, respectively. Interestingly, we observed one E. coli (HG4) isolate with both bla_NDM-1 and mcr-1 gene. Further analysis showed HG4 isolate has lesser survivability on the cotton swab, long lag phase and less biofilm production compared to colistin-sensitive isolates. Detection of E. coli with bla_NDM-1 and mcr-1 in this geographical region is an alarming signal for tourists, community, health workers and policymakers. Hence, it is utmost important to take appropriate measures to control the dissemination of antibiotic resistance gene in such pristine locations.