Background and objectives: Carbapenem-resistant and biofilm producing Enterobacteriaceaeare a major health problem. This study was carried to determine the antibacterial and antibiofilm activity of grape seed extract (GSE) against carbapenem-resistant and biofilm producing Enterobacteriaceae isolates. Methods: Antibiotics susceptibility patterns were detected by the disk diffusion method. carbapenem-resistant Enterobacteriaceae (CRE) isolates were screened by carbapenems disks and imipenem minimum inhibitory concentrations (MIC). The biofilm formation was detected by the microplate method. The carbapenemase genes were detected by PCR. The total polyphenolic content of GSE was determinate by Folin Ciocalteu technique. The antibacterial and antibiofilm effects of GSE were tested by the MIC and biofilm inhibitory concentration (BIC), respectively. Results: In this study, total phenolic content of extracted 1 gram of GSE was equivalent to 700 mg gallic acid. Eighteen non-duplicated CRE isolates were selected. All isolates were fosfomycin susceptible. Variable frequency of resistance to the other tested antibiotics was observed. The blaOXA-48 was the most common carbapenemase type. Among 18 isolates, 13 were biofilm producer while GSE inhibited CRE growth at 1024 µg/mL for 15 isolates and 2048 µg/mL for three isolates. Biofilm production was inhibited by GSE in 2000 µg/mL, 4000 µg/mL and 8000 µg/mL after 72 h incubation. Conclusion: The significant antibacterial and antibiofilm effects of GSE suggested GSE as a promising candidate for treatment of infections caused by these organisms