The application of solid-phase denitrification (SPD) as a system for treating wastewater with biofilms underscores the necessity of developing efficient acclimation strategies to cultivate resilient biofilms. This research examined how N-acyl homoserine lactones (AHLs) influence the characteristics and development of biofilms within a PHBV-supported SPD system. By incorporating five distinct AHLs into the SPD setup, it was found that the addition of 3-oxo-C14-HSL AHL significantly enhanced nitrate removal efficiency. Conversely, the introduction of exogenous C8-HSL and C14-HSL AHLs appeared to hinder the removal of pollutants. The quorum-sensing (QS) mechanism, mediated by AHLs, identified 3-oxo-C14-HSL as the pivotal AHL governing the production of tightly bound extracellular proteins (TB-PN) in the SPD apparatus. The introduction of exogenous 3-oxo-C14-HSL AHL proved to be instrumental in promoting the synthesis of amino acids akin to tryptophan, as well as in the formation of humic substances. This finding highlights the significant impact that this specific N-acyl homoserine lactone (AHL) can have on biological processes. Through the use of confocal laser scanning microscopy (CLSM), it was documented that the QS system, which is mediated by 3-oxo-C14-HSL, plays a crucial role in the formation of bioaggregates. This phenomenon is further characterized by an observable increase in the thickness of biofilms during biological denitrification processes. These findings underscore the importance of AHL-mediated QS in regulating the properties and development of biofilms within SPD systems. The insights gained from this study contribute significantly to the understanding of quorum-sensing mechanisms, thereby enhancing the theoretical framework surrounding this field of research. Furthermore, the practical implications of the study provide valuable recommendations for improving biofilm acclimation strategies in SPD applications, ensuring more effective and efficient biological treatment processes.