Balanced bacterial biofilm communities help to maintain host health. Disturbance of such balance can lead to bacterial dysbiosis and pathogenesis. However, complex and dynamic bacterial interactions within the biofilm communities are poorly understood. In this study, we used a dual-species biofilm consisting of the periodontal pathogen Aggregatibacter actinomycetemcomitans, and a commensal Streptococcus parasanguinis to investigate bacterial interactions since the two organisms have been found to coexist during the development of localized aggressive periodontal disease. We report that A. actinomycetemcomitans promoted biofilm formation of S. parasanguinis in vitro and in vivo. Protein profiling of S. parasanguinis co-cultured with A. actinomycetemcomitans revealed a significant decrease in the protein level of pyruvate oxidase(PoxL), an enzyme required for the generation of hydrogen peroxide (H2O2). Consistently, the H2O2 concentration was concurrently decreased. However, the complete removal of H2O2 impaired the biofilm formation. H2O2 at a low concentration range regulated by A. actinomycetemcomitans enhanced the biofilm formation. These results demonstrate that A. actinomycetemcomitans promotes the S. parasanguinis biofilm formation through modulating the production of H2O2 by fine-tuning the expression of poxL, indicating that H2O2 functions as a signaling molecule. Taken together, this report revealed a previously unknown bacteria-bacteria interaction mechanism.