Background Acinetobacter spp are important opportunistic pathogens responsible for nosocomial infections. Objectives: detection of EsβL and carbapenemase, the ability of biofilm formation and their relation to antimicrobial resistance. Methodology: A total of 230 clinical samples from patients admitted to Menoufia University Hospitals were obtained. Acinetobacter spp were identified by standard microbiological methods and Vitek-2 system. The antibiogram of Acinetobacter isolates was tested by the modified Kirby Bauer disk diffusion method, detection of extended-spectrum β-lactamases and carbapenemase by EsβL NDP and CANP tests. Biofilm production was detected by modified Congo red agar and PCR. Results Acinetobacter spp. represented (20.8%) of all the collected isolates. Vitek-2 system showed that the predominant spp. was Acinetobacter baumannii complex (80%). Acinetobacter isolates were highly resistant to cefepime and tobramycin (90% for each), ceftriaxone (88%), piperacillin, and ampicillin –sulbactam (86% for each), piperacillin- tazobactam (84%) and tetracycline (78%). About 64% and 68% of the Acinetobacter isolates were susceptible to tigecycline and colistin respectively. The sensitivity of EsβL NDP for detection of EsβL producing Acinetobacter isolates was 93.8 %. The Carba NP and carbAcineto NP tests detect carbapenemse production in 6% and 56% of Acinetobacter isolates respectively. Biofilm production was found among 56% isolates by MCRA method, while conventional PCR showed fimH and CsgA genes among 60% and 18% isolates respectively. Conclusion: Acinetobacter spp are serious nosocomial pathogens as they can produce ESβL and carbapenemase, and produce biofilm that is related to their antimicrobial resistance. Therefore, their adequate prevention and control is imperative.