Carbapenem-resistant Gram-negative bacilli (GNB) are a concern in the Middle East and worldwide. Simple screening methods have been sought to detect carbapenemase producers to determine appropriate therapeutic measures and implement infection control interventions. In this study, we evaluated the efficiency of agar disc diffusion, commercial combined disc test (Rosco), and carbapenem MIC determination in comparison to molecular detection of carbapenemase genes among 82 carbapenem non-susceptible Enterobacteriaceae (CNSE) and 37 Acinetobacter/Pseudomonas isolates. The blaOXA-48, blaNDM, blaNDM/OXA-48, and blaIMP were detected in 68 out of 82 CNSE isolates. All of the Acinetobacter baumannii isolates were positive for the blaOXA-51 (n = 23), of those some were positive for blaOXA-48 (n = 13) and blaNDM (n = 3). Sensitivities and specificities of combined disc test for detection of blaNDM and blaOXA-48 carrying Enterobacteriaceae isolates were 92.5% and 100%, and 58.5% and 100%, respectively, while those for Acinetobacter/Pseudomonas isolates were 100%, 81.8% and 96.2%, 89%, respectively. While carbapenem MIC values had excellent concordance with phenotypic combined disc test for detection of blaOXA-48 producers (area under curve > 90%), only ertapenem MIC's could precisely detect blaOXA-48 PCR-positive Enterobacteriaceae isolates (AUC 70%, sensitivity 70%, specificity 50%). The phenotypic commercial test showed excellent sensitivity for detection of blaNDM producers, but had poor sensitivity for blaOXA-48-producing Enterobacteriaceae. Ertapenem MIC values had low sensitivity and specificity for detection of the blaOXA-48-carrying Enterobacteriaceae. This is the first report of A. baumannii isolates co-harbored the blaOXA-48/blaNDM carbapenemases from Iran.