Background: Enterobacter spp., which are able to carry a number of antibiotic-resistance genes, can cause a variety of infections especially in immune-compromised individuals and patients from intensive care units (ICUs). Objectives: To assess the prevalence of Enterobacter spp. in Menoufia University Hospitals, and to investigate the relation between antimicrobial susceptibility patterns and biofilm production. Methodology: A total 296 clinical samples from patients admitted to Menoufia University Hospitals. Enterobacter spp. were identified by standard microbiological methods and Vitek-2 system. All Enterobacter isolates antibiogram was tested by the modified Kirby Bauer disk diffusion method, and for extended-spectrum β-lactamases and metallo-β-lactamase production. Biofilm production was detected by congo red agar, modified congo red agar methods and PCR. Results: Enterobacter spp. represented 17.3% of all the collected nosocomial isolates. Vitek-2 system showed that the predominant spp. was Enterobacter aerogenes (44%). Enterobacter isolates were resistant to amoxicillin (100%), doxycycline (82%) and gentamycin (76%). The rates of resistance to ceftriaxone, cefoxitin, cefepime, and amikacin were 64%, 72%, 60% and 70% respectively. Half of Enterobacter isolates were sensitive to piperacillin/tazobactam, meropenem, ciprofloxacin, ofloxacin and norfloxacin while 84% were sensitive to chloramphenicol. Production of ESβLs and MβL was found among 28% and 22% of isolates respectively. Biofilm production was found among 50% by CRA method and 56% by MCRA method, while conventional PCR showed fimH gene among 58% of Enterobacter isolates. Conclusion: Enterobacter spp. are serious nosocomial pathogens as they can produce ESβLs and carbapenemase, and produce biofilm that is related to their antimicrobial resistance. Therefore, their adequate prevention and control is imperative.