Bacteria are capable of evolving high doses of the drug in various infections by forming biofilms. Perhaps, biofilm regulator genes have different frequencies in β-lactam producing non-fermentative Gram-negative Bacilli (NFGNB). In this study, we investigated the role of biofilm operons of Pseudomonas aeruginosa and Acinetobacter baumannii on the prevalence of different β-lactamase enzymes. One-hundred twenty (120) nosocomial NFGNB isolates were collected from different clinical samples of patients. PCR method was used for the amplification of resistance genes. Isolates were collected, including 50 isolates (41.66%) of P. aeruginosa and 70 isolates (58.33%) of A. baumannii. The distribution of ESBL, AmpC, KPC, and MBL β-lactamase enzymes in P. aeruginosa and A. baumannii isolates were 64%, 58%, 38%,44%, and 57.14%, 60%, 32.85%, 34.28%, respectively. The frequency of csuABC, pgaABC operon in A. baumannii were as follows:  pgaA (45.71%), pgaB (32.85%), pgaC (42.85%), csuA (34.28%), csuB (32.85%), csuC (41.42%), and ompA (38.57%). Further, the prevalence of pslABC and pelABC operons in P. aeruginosa isolates were as follows:  pslA (58%), pslB (58%), pslD (60%), pelA (64%), pelB (38%), pelC (44%), and algD (68%). This study revealed that the abundance of biofilm regulator genes in NFGNB strains is affected by different β-lactamase enzymes.