Objective: The purpose of the present study was to identify the fermenting and non-fermenting Gram-negative bacteria from the tertiary care hospital. Methods:The conventional method of identification by biochemical analysis and antibiotic susceptibility test was performed by Kirby-Bauer disc diffusion method. Furthermore, analysis of microbes was done by Vitek-2.Results: 424 strains of lactose fermenting Gram-negative bacilli (LFGNB) and non-LFGNB (NLFGNB) were isolated from 3097 clinical samples. From the total LF bacteria, Escherichia coli was the predominant isolate accounting for 50.94% specimens, followed by Klebsiella pneumoniae 27.59% and Enterobacter 0.47%. From the total NLFGNB, Acinetobacter baumannii was the predominant isolate accounting for 12.73% specimens followed by Pseudomonas aeroginosa 6.13%, other isolates were Stenotrophomonas maltophilia 1.17%, Burkholderia cepacia 0.94%. In the present study, male were more infected than female. The study also showed that LF bacteria were more infectious than non LF bacteria and isolates were from urine samples. Conclusion:Both NLFGNB and LFGNB Gram-negative bacilli were found to be major contaminants, and are important pathogenic bacteria causing a wide range of infections in the tertiary care hospital.