[Objective] To explore the influence of Acinetobacter pittii LSQ 3 on the biofilm formation of Bacillus velezensis LSQ 19 and analyze the genomic characteristics of strain LSQ 3. [Methods] The effect of the cell-free supernatant (CFS) of LSQ 3 on the biofilm formation of LSQ 19 was analyzed by crystal violet staining, cell surface property analysis, the phenol-sulfuric acid method, XTT reduction assay, and scanning electron microscopy (SEM). Whole-genome sequencing was employed to determine the taxonomic status of strain LSQ 3, and the biosynthetic gene clusters for secondary metabolites were predicted based on the whole-genome data. [Results] The CFS of strain LSQ 3 significantly inhibited the biofilm formation of strain LSQ 19, and the volume ratio of 10 µL bacterial suspension to 190 µL CFS was determined as the minimum inhibitory concentration (MIC). Compared with the control, the CFS of strain LSQ 3 at MIC significantly reduced the surface hydrophobicity, adhesion, extracellular polymeric substances (EPS) production rate, and biofilm metabolic activity, while significantly improving the self-aggregation ability of LSQ 19 cells. In the presence of the CFS at MIC, LSQ 19 failed to form a biofilm on the glass surface. Strain LSQ 3 was identified as A. pittii based on whole-genome sequencing data. Its genome size was 3 939 365 bp, with the G+C content of 38.82% and 3 601 DNA coding sequences. The genome contained multiple genes involved in biofilm formation and virulence factors. The antiSMASH analysis showed that the genome of strain LSQ 3 contained seven biosynthetic gene clusters for secondary metabolites. [Conclusion] The CFS of A. pittii LSQ 3 can inhibit the biofilm formation of B. velezensis LSQ 19. This study provides a theoretical basis and reference for the construction of synthetic bacterial communities from the perspective of biofilms.