Physicochemical Characterization, Antimicrobial and Antibiofilm Activities of Thymus vulgaris and Rosmarinus officinalis Essential Oil Nanoemulsions with Potential Mouthwash Applications

Background/Objectives: In this study, nanoemulsions containing Thymus vulgaris (TV) and Rosmarinus officinalis (RO) essential oils (EOs) were prepared and characterized, and their antibacterial, antifungal, and antibiofilm activities were evaluated against some oral pathogens. Methods: Three nanoemulsions containing 5% TV-EO (F1), 5% RO-EO (F2), and a combination of 2.5% TV-EO and 2.5% RO-EO (F3) were prepared by emulsification followed by high-speed homogenization. Results: Gas chromatography–mass spectrometry coupled with flame ionization detection (GC-MS-FID) analysis revealed linalool (79.51%) as the major constituent of TV-EO and eucalyptol (58.13%), together with camphor (10.80%), as the predominant compounds of RO-EO. All nanoemulsions exhibited nanosized droplets, low polydispersity index (PDI) values (<0.3), and moderate to high colloidal stability. Transmission electron microscopy (TEM) analysis revealed predominantly spherical droplets in all formulations (F1–F3). All formulations exhibited antimicrobial activity against Streptococcus mutans, Lactobacillus acidophilus, Enterococcus faecalis, and Candida albicans. Among the tested formulations, F2 showed the strongest antibacterial activity, whereas F3 exhibited the highest antibiofilm activity against mature S. mutans biofilms, achieving approximately 80% inhibition, comparable to chlorhexidine (CHX). Although F3 possessed the smallest droplet size, its antimicrobial activity was lower than that of F2, suggesting that biological activity was influenced not only by droplet size but also by EO composition. Conclusions: These findings demonstrate the potential of TV-EO and RO-EO nanoemulsions as promising natural alternatives for mouthwash formulations and highlight the importance of EO composition in determining antimicrobial and antibiofilm efficacy.