| Summary: | (1) Background: <i>Acinetobacter baumannii</i> is well known as a causative agent of severe hospital-acquired infections, especially in intensive care units. The present study characterised the genetic traits of biofilm-forming carbapenem-resistant <i>A. baumannii</i> (CRAB) clinical isolates. Additionally, this study determined the prevalence of biofilm-producing <i>A. baumannii</i> isolates from a tertiary care hospital and investigated the association of biofilms with the distribution of biofilm-related and antibiotic resistance-associated genotypes. (2) Methods: The 995 non-duplicate <i>A. baumannii</i> isolates were identified, and their susceptibilities to different antibiotics were determined using the disk diffusion method. Using the modified microtiter plate assay, the CRAB isolates were investigated for their biofilm formation ability. Hemolysin and protease activities were determined. CRABs were subjected to polymerase chain reaction (PCR) assays targeting <i>bla</i><sub>VIM</sub>, <i>bla</i><sub>NDM</sub>, <i>bla</i><sub>IMP</sub>, <i>bla</i><sub>OXA-23-like</sub>, <i>bla</i><sub>OXA-24-like</sub>, <i>bla</i><sub>OXA-51-like</sub>, <i>csu</i>E and <i>pga</i>B genes. Individual CRAB isolates were identified for their DNA fingerprint by repetitive element sequence-based (REP)-PCR. (3) Results: Among all <i>A. baumannii</i> isolates, 172 CRABs were identified. The major antibiotic resistance gene among the CRAB isolates was <i>bla</i><sub>OXA-51-like</sub> (100%). Ninety-nine isolates (57.56%) were biofilm producers. The most prevalent biofilm gene was <i>pga</i>B (79.65%), followed by <i>csu</i>E (76.74%). Evidence of virulence phenotypes revealed that all CRAB exhibited proteolytic activity; however, only four isolates (2.33%) were positive for the hemolytic-producing phenotype. REP-PCR showed that 172 CRAB isolates can be divided into 36-DNA fingerprint patterns. (4) Conclusions: The predominance of biofilm-producing CRAB isolates identified in this study is concerning. The characterisation of risk factors could aid in controlling the continual selection and spreading of the <i>A. baumannii</i> phenotype in hospitals, thereby improving patient care quality.
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