Peri-implantitis associated with dental implants shares characteristics with destructive periodontal diseases. Both conditions are multifactorial and strongly correlated with the presence of microorganisms surrounding the prostheses or natural dentition. This study aimed to evaluate the antimicrobial activity and toxicity of a mucoadhesive hydrogel functionalized with aminochalcone (HAM-15) against Aggregatibacter actinomycetemcomitans, Fusobacterium periodonticum, Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia, and Candida albicans. Various experiments were conducted to determine the minimum inhibitory concentrations (MIC) and minimum bactericidal/fungicidal concentrations (MBC/MFC), as well as the antibiofilm potential and toxicity in human gingival fibroblasts and a G. mellonella animal model. Infection and treatment studies were also performed in G. mellonella. The results demonstrated that both aminochalcone (AM-15) and the aminochalcone-functionalized hydrogel (HAM-15) exhibited antimicrobial activity, with MICs ranging from 7.8 to 31.2 μg/mL for the tested strains. Treatment with HAM-15 at 300 μg/mL reduced the monospecies biofilm of C. albicans and P. gingivalis by 7 log