Introduction: Non-fermentative Gram-negative bacilli (NFGNB) are an important cause of healthcare-associated infections, and theyare primarily opportunistic pathogens.Aim and objectives: To isolate and characterize the non-fermenting Gram-negative bacilli (NFGNB) from various clinical samples ina tertiary care center. To detect the virulence markers and drug resistance using molecular methods.Material and methods: Non-fermentative Gram-negative bacilli (NFGNB) from various clinical samples were identified using standardbacteriological identification methods. Conventional PCR for detection of MBLs genes of P. aeruginosa such as blaIMP, blaVIM, blaNDMand a virulence marker, Exo S gene and also MBL genes of A. baumannii such as blaIMP, blaVIM, blaNDM, blaOXA-23, blaOXA-58, and avirulence marker, OmpA gene.Results: Out of 104 NFGNB isolates Pseudomonas aeruginosa was the most common bacteria comprising 62% (n=65), followed byAcinetobacter baumannii 25% (n=26), Acinetobacter lwoffii 4% (n=4), Pseudomonas flourescens 3% (n=3), Burkholderia cepacia 3%(n=3), Stenotrophomonas maltophilia 1% (n=1), Myroides odoratimimus 1% (n=1), and Rhizobium radiobacter 1% (n=1). The resultof P. aeruginosa showed that the blaVIM gene was expressed in 25 %(n=2), the blaNDM gene was expressed in 50 %(n=4) and blaIMPwas found in 25 %(n=2). For A. baumannii, the blaNDM gene was expressed in 33% (n=6), blaOXA-23 gene was expressed in 78%(n=14), blaOXA-58 gene was expressed in 56% (n=10) with no expression of blaIMP and blaVIM genes.Conclusion: In our study we report the increased prevalence of MBL genes in P. aeruginosa and A. baumannii. This highlights theimportance of accurate identification of MBL producers and judicious use of carbapenems. Early identification and continuedsurveillance of these multidrug resistant organisms will help to prevent their spread in a hospital environment.