Oral infections, especially in immunocompromised patients, represent a growing therapeutic challenge due to antimicrobial resistance and the limited availability of effective topical alternatives. This study aimed to develop and characterize bioactive chitosan (CH)-based films incorporated with copaiba oleoresin (COR) to contribute to the treatment of oral diseases.

Polymeric films containing CH incorporated with different COR concentrations (0.5%, 1%, and 3.5%) were formulated and characterized using Raman and FT-IR spectroscopy. Moisture, swelling, and solubility were evaluated. Cytotoxicity was tested in J774A.1 cells (MTT assay). COR was previously analyzed by GC-MS and subsequently tested for Minimum Inhibitory Concentration (MIC) and Minimum Microbicidal Concentration (MMC) against 17 pathogens of oral relevance. The CH–COR combination was evaluated by the checkerboard method (Fractional Inhibitory Concentration Index, FICI) and biofilm inhibition assay.

Spectroscopic analysis confirmed the presence of bioactive compounds with known antimicrobial properties, such as β-caryophyllene and diterpenic acids. COR exhibited MIC values between 26.04–46.87 µg/mL, and CH between 0.1–0.8 mg/mL. The combination demonstrated a synergistic effect against Streptococcus oralis(ATCC 10557) and an additive effect against other strains tested, with bactericidal action and satisfactory inhibition of biofilm formation, with values close to azithromycin control. The films presented moisture content ranging from 15.24% to 20.23%, with lower humidity at higher COR concentrations. Swelling was higher during the first hours, suggesting an ideal dressing replacement around 10 hours. Solubility ranged from 94.1% to 96.5%, which may compromise sustained release. The cytotoxicity of the films was inconclusive due to physical interference in the assay; however, isolated COR was safe and promoted cell growth, and CH demonstrated biocompatibility.

CH–COR films exhibited promising physicochemical properties, good initial absorption, high solubility, and antimicrobial potential due to the active compounds incorporated into the matrix. The combination showed synergism against oral pathogens and effective biofilm inhibition, making it a promising candidate for managing oral infections. Further studies are needed to optimize evaluation methods and ensure clinical safety and efficacy.