Thyme oil-in-water nanoemulsions stabilized by a nonionic surfactant (Tween 80, T80) were prepared as potential antimicrobial delivery systems (pH 4). The nanoemulsions were highly unstable to droplet growth and phase separation, which was attributed to Ostwald ripening due to the relatively high water solubility of thyme oil. Ostwald ripening could be inhibited by incorporating ≥75% of corn oil (a hydrophobic material with a low water solubility) into the nanoemulsion droplets. The electrical characteristics of the droplets in the nanoemulsions were varied by incorporating ionic surfactants with different charges after homogenization: a cationic surfactant (lauric arginate, LAE) or an anionic surfactant (sodium dodecyl sulfate, SDS). The antifungal activity of nanoemulsions containing positive, negative, or neutral thymol droplets was then conducted against four strains of acid-resistant spoilage yeasts: Zygosaccharomyces bailli, Saccharomyces cerevisiae, Brettanomyces bruxellensis, and Brettanomyces naardenensis. The antifungal properties of the three surfactants (T80, LAE, SDS) were also tested in the absence of thymol droplets. Both ionic surfactants showed strong antifungal activity in the absence of thymol droplets, but no antimicrobial activity in their presence. This effect was attributed to partitioning of the antimicrobial surfactant molecules between the oil droplet and microbial surfaces, thereby reducing the effective concentration of active surfactants available to act as antimicrobials. This study shows oil droplets may decrease the efficacy of surfactant-based antimicrobials, which has important consequences for formulating effective antimicrobial agents for utilization in emulsion-based food and beverage products.