Objectives: The non-lactose fermenting Gram-negative bacilli (NLF-GNB) are notorious pathogens reportedly acquiring multiple drug resistance alarmingly and emerging as a public health threat globally. This study was conducted to isolate and identify these pathogens from clinical samples received routinely in our Bacteriology Laboratory and to analyze their antibiotic susceptibility patterns. Methods: In this cross-sectional study, the first 100 NLF-GNB strains isolated consecutively from 1218 clinical samples were included through convenience sampling. The samples were processed using standard microbiological techniques. Results: The most common isolate was Pseudomonas aeruginosa followed by Acinetobacter spp, Proteus spp, Shigella spp, Salmonella typhi, Providencia spp., and Morganella spp. P. aeruginosa and Acinetobacter spp. isolates were found to exhibit high susceptibility toward Colistin and Imipenem. Proteus spp. exhibited high sensitivity toward Imipenem, Aminoglycosides, Ceftazidime, and Cefepime. All Providencia isolates were susceptible to Amikacin, Cefepime, and Ceftriaxone. The only isolate of Morganella spp. was found to be susceptible to Amikacin, Cefepime, Ceftazidime, Piperacillin tazobactam, Ciprofloxacin, Imipenem, and Aztreonam. Shigella isolates exhibited very high susceptibility toward Imipenem followed by Gentamicin and Ceftazidime. All the isolates of S. typhi exhibited susceptibility toward Imipenem, Piperacillin tazobactam, Ceftazidime, Ceftriaxone, Cefoperazone sulbactam, and Chloramphenicol. 24% of test isolates were found to be Multidrug resistant. Conclusion: Antimicrobial surveillance is needed to implement appropriate timely interventions to restrict the spread of multidrug-resistant clones. Strict infection prevention and control practices, with judicious antibiotic prescription policy, may help in tackling this problem by obviating the selection pressure.