The aim of this study was to identify carbapenemase genes distributed among carbapenem-resistant Gram-negative bacteria (CR-GNB), previously collected from medical samples from different sites in King Faisal Medical Complex Hospital (KFMC), Taif, Saudi Arabia, and to determine the ability of these CR-GNB isolates to biofilm formation. The bacterial samples, collected from patients at FMC from November 2021 to April 2022 (6 months), were screened for carbapenem resistance (CR) by Phoenix System. Further, the detection of carbapenemase producer (CP) genes was performed using the XpertCarba-R molecular method. Overall, 236 clinical samples were determined as CR-GNB from 167 CR patients. The real-time PCR results show that 40.7% (68/167) of the CR isolates were positive for bla_NDM and 33.5% (56/167) were positive for bla_OXA while no isolates were positive for bla_KPC, bla_VIM and bla_IMP which mean41.3% (69/167) of the CR isolates didn't have any carbapenemase genes. There was a significant association between the type of carbapenem resistance genes within various CRorganisms, antibiotypes categories, and hospital service wards. There were three distinct groups of CP isolates circulating in KFMC. The first group of strains was positive for bla_OXA (30, 18.6%). The second group of strains was positive for bla_NDM 42 (25.1 %). The third group of isolates was positive for both bla_NDM and bla_OXA (26, 15.6%). Most CR Klebsiella spp. isolates were strong biofilm former (74/99, 74.7%) while lower numbers of CR Klebsiella spp. formmoderate biofilm (25/99, 25.3%). Also, the strong biofilms were the most common among CR Pseudomonas spp. (18/23, 78.3%), Acinetobacter baumannii (25/32, 78.1%), and other organisms (13/13, 100%). Weak biofilm just appeared in two isolates (8.7%) of Pseudomonas spp.It is clear that biofilm formation ability didn't distribute randomly in the various CR organisms and antibiotype categories. Carbapenemase-producing ability and biofilm formation ability are the most virulent factors of CR-GNB, the increase of carbapenemase-producing organisms in biofilm-producing isolates of CR-GNB is considered a serious alert