In recent years, infections frequently attributed to Pseudomonas aeruginosa and Acinetobacter baumannii have been seen not only in ICU settings but also in trauma patients following natural disasters, as well as in individuals with pre-existing conditions within the community. These non-fermenting bacteria are known to cause hospital-acquired bloodstream infections, especially in patients who are debilitated or immunocompromised. All non-fermenting Gram-negative bacteria (NFGNB) isolates from different clinical samples received in the clinical microbiology laboratory from outpatients and inpatients at Mahatma Gandhi Medical College and Hospital, Jaipur, Rajasthan, were included in the study. Routine microscopy of these samples was conducted. Gram staining was realized for all samples except for urine. All clinical samples were inoculated on blood agar and MacConkey agar and were incubated at 37°C for 18–24 hours. Colony characteristics were observed. All the organisms that produced pale or colorless colonies on MacConkey agar and also showed GNB on Gram staining of the colonies were considered to be NFGNB and further identified by the VITEK 2 compact system. Antimicrobial susceptibility testing of the isolated non-fermenting Gram-negative bacteria was conducted using the Kirby-Bauer disc diffusion method. In the study, it was reported that the majority out of 879 (100%) isolates of NFGNB were P. aeruginosa 415 (47.21%), followed by Ac. baumannii 380 (43.23%), and the majority of specimens were from endotracheal (ET) (30.60%), followed by pus swab (19.11%), blood (16.15%) and urine (10.01%) samples. The majority of isolates were the males in the age group of 61-70, i.e.121 (13.76%), followed by the age group 41-50, i.e., 113 (12.85%). P. aeruginosa was most commonly isolated from pus swabs (13.42%), while Ac. baumannii was isolated most commonly from ET (21.4%). Based on this study, it can be concluded that as antibiotic resistance rises, treating infections caused by NFGNB will become increasingly challenging unless proactive measures are implemented and new antibiotics are developed. To curb the spread of resistant strains of Pseudomonas aeruginosa and Acinetobacter, it is crucial to enforce stringent infection control practices, ensure collaboration between clinicians and laboratory personnel regarding antibiotic use, and adhere to strict hospital hygiene protocols.