IntroductionPseudomonas aeruginosais an opportunistic pathogen, which causes healthcare-associated infections in immunosuppressed patients. They exhibit resistance to multiple classes of antibiotics via various mechanisms such as the over-expression of efflux pumps, decreased production of the outer membrane protein (D2 porin), over-expression of the chromosomally encoded AmpC cephalosporinase, modification of drugs, and mutation(s) at the target site of the drug. The bacteria also develop antibiotic resistance through the acquisition of resistance genes carried on mobile genetic elements. Limited data on phenotypic as well as genotypic characterization of MDRP. aeruginosain Nepal infers the needs for this study. This study was carried out to determine the prevalence rate of metallo-β-lactamase (MBL-producer) as well as colistin resistant multidrug resistant (MDR)P. aeruginosain Nepal and also to detect MBL, colistin resistance, and efflux pump encoding genes i.e.blaNDM−1,mcr-1andMexBrespectively in MDRP. aeruginosaisolated from clinical samples.Methods/methodologyA total of 36 clinical isolates ofP. aeruginosawere collected. All bacterial isolates were phenotypically screened for antibiotic susceptibility using Kirby Bauer Disc Diffusion method. All the multidrug resistantP. aeruginosawere phenotypically screened for MBL producer by Imipenem-EDTA combined disc diffusion test (CDDT). Similarly, MIC value for colistin was also determined by broth microdilution method. Genes encoding carbapenemase (blaNDM−1), colistin resistant (mcr-1) and efflux pump activity (MexB) were assayed by PCR.ResultsAmong 36P. aeruginosa, 50% were found to be MDR among which 66.7% were found to be MBL producer and 11.2% were found to be colistin resistant. Among MDRP. aeruginosa, 16.7%, 11.2% and 94.4% were found to be harbouringblaNDM−1,mcr-1andMexBgenes respectively.ConclusionIn our study, carbapenemase production (encoded byblaNDM−1), colistin resistant enzyme production (encoded bymcr-1), and expression of efflux pump (encoded byMexB) are found to be one of the major causes of antibiotic resistance inP. aeruginosa. Therefore, periodic phenotypic as well as genotypic study in Nepal onP. aeruginosawould provide the scenario of resistance pattern or mechanisms inP. aeruginosa. Furthermore, new policies or rules can be implemented in order to control theP. aeruginosainfections.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12866-023-02906-w.