Multidrug‐resistant (MDR) Acinetobacter baumannii poses a significant therapeutic challenge due to its resistance to multiple antibiotics and its ability to form biofilm. This study aimed to characterize MDR A. baumannii isolates for their biofilm‐forming capabilities and the presence of common biofilm‐related genes at a tertiary care university hospital in Nepal. In addition, it assessed the efficacy of various compounds, particularly essential oils, in inhibiting biofilm formation. Identification and antibiotic sensitivity testing of A. baumannii isolates from clinical specimens were conducted according to the guidelines of the American Society for Microbiology. Isolates were screened for motility profiles, biofilm production in a microtiter plate assay, and the presence of biofilm‐related gene(s) by conventional polymerase chain reaction. The ability of cinnamaldehyde, ethylenediaminetetraacetic acid (EDTA), Tween 80, amino acids (glycine and glutamic acid), and natural plant extracts to inhibit biofilm formation was also tested using the microtiter plate system. Out of the total 200 A. baumannii isolates, 195 were MDR, with 192 able to produce biofilms. Among them, 83.1% were strong biofilm producers. In this study, 42.0% and 66.2% of the isolates exhibited twitching motility and surface‐associated motility, respectively. Thirty MDR A. baumannii isolates from medical devices contained biofilm‐related genes csuE, ompA, bap, and blaPER−1, in 90.0%, 53.3%, 46.6%, and 26.6% of strains, respectively. Cinnamaldehyde (0.875 mg/mL) was the most effective compound, inhibiting biofilm formation by 77.3%, followed by ethanolic extract of onion (77.2%), 0.5% Tween 80 (76.8%), and essential oil of ginger (70.8%). The majority of A. baumannii clinical isolates were strong biofilm producers and often possessed the biofilm‐related genes csuE and ompA. Essential oils at 200 mg/L, along with Tween 80, were the most effective (≥ 67%) at inhibiting the formation of biofilms. These findings help to understand biofilm production and provide valuable insights into MDR A. baumannii isolates in this clinical setting.