Dental biofilm (DB) is a complex microbial structure associated with caries and periodontal disease, representing a major focus of oral microbiology research. Bacteria such as Streptococcus mutans and Streptococcus sobrinus are cariogenic due to their acid production, which promotes enamel demineralization. Preventing DB formation is challenging given its structural and microbial complexity, along with genetic and environmental factors. In this context, natural products have been investigated as alternatives to synthetic antimicrobials due to their lower toxicity, better biocompatibility, and reduced cost. Punica granatum (pomegranate) is known for its antimicrobial activity, attributed to phenolic compounds such as tannins and flavonoids. Although the fruit, seeds, and flowers have been studied, the leaves of P. granatum remain poorly explored. This study aimed to develop a hydroalcoholic extract from pomegranate leaves (HPE) and evaluate its antibacterial and antibiofilm activity against oral microorganisms.

P. granatum leaves were collected, macerated in 50% hydroalcoholic solution, and evaporated. The HPE was tested against bacterial and fungal strains relevant to dentistry using MIC and MBC tests according to CLSI (2022). Activity against S. mutans and S. sobrinus biofilms was evaluated through adhesion and disruption assays. Characterization was performed using Raman spectroscopy and AFM. Cytotoxicity was tested on J774A1 cells (MTT assay).

The HPE showed significant antimicrobial activity, with MICs ranging from 0.39 to 6.25 µg/mL for bacteria and 1.56 µg/mL for yeasts. It demonstrated bactericidal activity against most strains. It inhibited S. sobrinus adhesion by up to 66.27% (2 × MIC) and showed biofilm-disrupting efficacy similar to azithromycin. The extract displayed low cytotoxicity and homogeneous structure, with viability higher than the control at all concentrations.

The HPE exhibited relevant antimicrobial and antibiofilm potential, with low toxicity and high efficacy against oral pathogens. Although the results are promising, further studies are needed to elucidate its mechanisms of action and validate its clinical application as a complementary therapy in dentistry.