<p>Two-component signaling pathways allow bacteria to sense and respond to environmental changes, yet the sensory mechanisms of many remain poorly understood. In the pathogen <i>Vibrio cholerae</i>, the DbfRS two-component system controls the biofilm lifecycle, a critical process for environmental persistence and host colonization. Here, we identified DbfQ, a small periplasmic protein encoded adjacent to <i>dbfRS</i>, as a direct modulator of pathway activity. DbfQ directly binds the sensory domain of the histidine kinase DbfS, shifting it toward phosphatase activity and promoting biofilm dispersal. In contrast, outer membrane perturbations, caused by mutations in lipopolysaccharide biosynthesis genes or membrane-damaging antimicrobials, activate phosphorylation of the response regulator DbfR. Transcriptomic analyses reveal that DbfR phosphorylation leads to broad transcriptional changes spanning genes involved in biofilm formation, central metabolism, and cellular stress responses. Constitutive DbfR phosphorylation imposes severe fitness costs in an infection model, highlighting this pathway as a potential target for anti-infective therapeutics. We find that <i>dbfQRS</i>-like genetic modules are widely present across bacterial phyla, underscoring their broad relevance in bacterial physiology. Collectively, these findings establish DbfQ as a new class of periplasmic regulator that influences two-component signaling and bacterial adaptation.</p>