One of the major factors contributing to drug resistance in Acinetobacter nosocomialis infections is biofilm development, which is facilitate by quorum-sensing (QS) systems. Quorum sensing by the LuxI and LuxR homologues, AnoI and AnoR, in A. nosocomialis plays a role in biofilm formation and motility of this pathogenic bacterium. The aim of this study was to evaluate the effects of exogenous N-acyl-homoserine lactones (AHLs) on the regulation of biofilm and motility of A. nosocomialis and anoI-deletion mutant. We found that anoR mRNA expression levels in the anoI-deletion mutant were increased in the presence of different types of AHLs compared with that in the absence of exogenous AHL. Among AHLs, C12-HSL appeared to exert the greatest stimulatory effect on biofilm formation and motility. Notably, the anoI-deletion mutant also exhibited significant decreases in expression of the biofilm- and motility-related genes, csuC, csuD and pilT, decreases that were attenuated by addition of exogenous AHLs. Combining the AHL C12-HSL with C6-HSL or C10-HSL exerted synergistic effects that restored the motility phenotype in the anoI-deletion mutant. Taken together, our data demonstrate that C12-HSL may act as an important signaling molecule in A. nosocomialis through regulation of biofilm formation and cell motility, potentially providing a new target for the control of A. nosocomialis infections.