BACKGROUND Non fermenting gram-negative bacilli (NFGNB) are a group of heterogenous, aerobic and non-sporing saprophytic bacteria, found as commensals in humans and other animals primarily causing opportunistic healthcare-associated infections. They are innately resistant to many antibiotics and are known to acquire resistance by various mechanisms. They pose a particular difficulty for the healthcare community because multidrug resistance is common and increasing among them and a number of strains have now been identified that exhibit pan drug resistance. This study was conducted to isolate and identify various non-fermenter gram negative bacilli (NFGNB), to study their antibiotic sensitivity pattern and their clinical significance from various clinical samples. METHODS A study was undertaken from March 2019 to February 2020 to isolate NFGNB from various clinical samples received for culture and sensitivity in the department of microbiology in a tertiary care hospital, Ahmedabad. Non lactose fermenting colonies on MacConkey agar plates were further processed by Vitek 2 to identify them and to study their antimicrobial susceptibility testing (AST). RESULTS A total of 2010 NFGNB were isolated from various clinical samples and their AST was evaluated by Vitek 2. Pseudomonas aeruginosa (52.7 %) and Acinetobacter baumannii (36.5 %) were the most common NFGNB isolated. Carbapenem resistance was 93 % for Acinetobacter species and 61 % for Pseudomonas species. CONCLUSIONS Accurate and rapid identification and antimicrobial susceptibility testing of NFGNB help in early initiation of appropriate antimicrobial therapy and proper management of patients thereby help in reducing emergence of MDR strains of NFGNB, mortality and overall hospital stay. KEYWORDS NFGNB – Non-Fermenting Gram-Negative Bacilli, Multidrug Resistance, Pan Drug Resistance, Carbapenem Resistance