Comparison of the Antimicrobial Activity of Mouthwashes Containing Tea Tree Oil, Cannabidiol (CBD), Spilanthol, and Chlorhexidine Digluconate Against Selected Bacterial and Yeast Strains: An In Vitro Study

(1) Background: The oral cavity is a complex ecological environment that integrates elements of both the digestive and respiratory systems, contributing to its extensive microbial diversity. Despite its effectiveness, chlorhexidine is associated with undesirable effects, such as mucosal irritation and tooth staining, which have prompted research into natural alternatives. This study aimed to compare the antimicrobial activity of mouthwashes containing tea tree oil (TTO) alone and in combination with cannabidiol (CBD) and spilanthol with that of a chlorhexidine digluconate-based mouthwash (CHX) against selected oral bacterial and fungal strains. (2) Methods: To assess the antimicrobial effects of the tested mouthwashes on reference microbial strains, the agar diffusion method was applied in accordance with the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST). In addition, the microdilution method using 96-well microtiter plates was employed to determine the minimum inhibitory concentration (MIC) of the tested substances. Microbial viability was further evaluated using the WST-based colorimetric Microbial Viability Assay Kit, in which the intensity of the produced WST-formazan dye is directly proportional to the number of viable cells. (3) Results: In the disc diffusion assay, inhibition zones measured after 24 h varied among the tested microorganisms, with the largest zones observed for CHX against Candida parapsilosis (19.63 mm) and Streptococcus pyogenes (16.7 mm). In the microdilution assay against Candida albicans, the MIC50 was achieved for preparations A and B at the highest tested concentrations (column 1), whereas for chlorhexidine (CHX), it was reached at lower concentrations (column 9). (4) Conclusions: ① All tested mouthwashes containing tea tree oil (TTO), either as a single active ingredient or in combination with cannabidiol (CBD) and spilanthol, demonstrated limited bacteriostatic and antifungal activity under the experimental conditions of this study. ② The chlorhexidine digluconate-based mouthwash exhibited significantly higher antibacterial and antifungal activity against all tested microbial strains compared to both the TTO-only mouthwashes and the formulation containing TTO combined with CBD and spilanthol.