Background: Bacterial resistance to beta-lactam is a major problem in all developed and developing countries. The genera of Klebsiella and Enterobacter are associated with opportunistic and nosocomial infections. All beta-lactamase genes can cause dissemination of resistance to beta-lactams. Objectives: The present study aimed to investigate the phenotypic detection of beta-lactamases in Enterobacter and Klebsiella species isolated from clinical specimens. Materials and Methods:This cross-sectional study was performed on 59 Klebsiella spp. and 49 Enterobacter spp. isolated from clinical samples. They were confirmed using API 20E. These bacteria were evaluated for the production of extended-spectrum beta lactamase (ESBL), metallo-beta-lactamase (MBL) (IMP-1), and the pAmpC and iAmpC enzymes. This was done using the Clinical and Laboratory Standards Institute (CLSI) method, 2-mercaptopropionic acid, the cefoxitin method, and the use of imipenem as an enzyme inducer, respectively. Mask-ESBL production was also identified, using different concentrations of 3-amino-phenyl boronic acid compound. Data were analyzed with SPSS version 22. Results: In total, 38 (64.4%) Klebsiella spp. and 41 (83.7%) Enterobacter spp. produced at least one type of beta-lactamase. AmpCproducing Enterobacter spp. (71.4%), and ESBL-producing Klebsiella spp. (42.4%) had the highest prevalence of beta-lactamase types in each genus. There were two bacteria in both types that were resistant to all antibiotics without producing any type of beta-lactamase. Conclusions: According to our findings, it is necessary to pay special attention to ESBL production in Klebsiella spp., while in Enterobacter spp., it is essential to search for AmpC production (chromosomal and plasmid). In addition, the genotypic evaluation of beta-lactamase variety in these bacteria may be necessary in different geographical areas.