: This study aimed to investigate the effects of the quorum-quenching enzyme -acyl-homoserine lactone (AHL)-lactonase Est816 on biofilm formation in subgingival plaque microbiota from participants with advanced periodontitis. : Subgingival plaque samples were collected from 30 adults with untreated Stage III or higher periodontitis and cultured anaerobically. Est816 was applied in vitro, with phosphate-buffered saline (PBS) serving as the control. Biofilm composition was analyzed via 16S rRNA sequencing, and alpha diversity metrics were assessed. Differential taxa abundance was assessed with the multivariate statistical software MaAsLin3. Biofilm morphology, biomass, and thickness were evaluated using scanning electron microscopy (SEM), crystal violet staining, and confocal laser scanning microscopy (CLSM). : Est816 significantly reduced microbial richness (Chao1 Index, = 0.031), biofilm biomass (64% reduction, < 0.001), and thickness (76% reduction, < 0.001) compared to controls. SEM revealed fragmented biofilm architecture in Est816-treated samples. : AHL-lactonase Est816 inhibited polymicrobial subgingival-plaque-derived biofilm formation while reducing species richness, phylogenetic diversity, and community evenness. These findings demonstrate Est816's potential as an adjunctive therapy for disrupting pathogenic biofilms in periodontitis.