Non-fermenting gram-negative bacteria (NFGNB) endeavouring as major pathogen in infectious disease, predominantly in urinary tract infection (UTI) and increased resistance in NFGNB are matter of concern. This study aimed to evaluate the frequency of NFGNB, antibiotics resistance pattern, plasmid profiling, and antibacterial efficacy of Moringa oleifera against NFGNB. NFGNB were isolated from clinically suspected UTI patients. Identification of isolates and their antibiotics sensitivity pattern were analyzed according to conventional method, and Vitek 2 automated system. Moreover, NFGNB were evaluated for biofilm production and presence of plasmid. Furthermore, antibacterial activity of Moringa oleifera was evaluated against NFGNB. P. aeruginosa (86.0%), and A. baumannii (10.0%) were the most frequent NFGNB followed by Providencia rettgeri 2.0%, Stenotrophomonas maltophilia 1.0%, myroides species 1.0%. 68.6% P. aeruginosa and 60.0% A. baumannii were biofilm producers whereas imipenem and meropenem were the most effective antibiotics. Isolated NFGNB showed multiple bands of plasmid. Furthermore, Moringa oleifera leaves extract showed antibacterial activity against tested NFGNB. MDR-NFGNB presents challenges in treatment and Moringa oleifera leaf extract may be used as an alternative medicine. However, the therapeutic role of specific ingredients present in extract needs further investigation and purification.