AimsThis study aimed to evaluate the antibiofilm potential of phytol and cefotaxime combinations (PCCs) against Acinetobacter baumannii and to elucidate the molecular mechanism of their antibiofilm potential through the transcriptomic approach. Methods and ResultsPhytol and cefotaxime combination(s) (PCC(s) [160gml(-1)+8gml(-1) for microbial type culture collection (MTCC) strain and 160gml(-1)+0.5gml(-1) for clinical isolate] effectively inhibited the A. baumannii biofilm formation. Additionally, light, confocal laser scanning and scanning electron microscopic analyses validated the antibiofilm potential of PCCs. Furthermore, PCCs treated A. baumannii cells showed a decreased level of hydrophobicity index compared to their respective controls. Fourier-transform infrared (FT-IR) spectra of exopolysaccharide matrix extracted from PCCs-treated A. baumannii cells showed a visible decrease in absorbance of polysaccharides, nucleic acids and protein regions compared to the spectra of untreated controls. In the blood sensitivity assay, the PCCs-treated A. baumannii plates showed reduced a number of bacterial colonies compared to their control plates. Reduced level of catalase production was also observed in the PCCs treatment compared to their controls. Transcriptomic analysis revealed the downregulation of bfmR, bap, csuA/B, ompA, pgaA, pgaC and katE biofilm virulence genes in both the A. baumannii strains on treatment with PCCs. ConclusionThe obtained results of this study indicate that PCCs have potent antibiofilm activity and downregulate the biofilm-related virulence genes expression in A. baumannii. Significance and Impact of the StudyTo the best of our knowledge, this is the pioneering study, which shows the antibiofilm effect of PCCs against A. baumannii along with their molecular mechanism. The antibiofilm effect of PCCs could be a successful strategy for eradicating infections related to A. baumannii biofilms in nosocomial settings.