The use of non-Saccharomyces yeasts, such as Lachancea thermotolerans, is gaining increasing interest in winemaking due to their capacity to enhance the sensory and chemical profile of wines, notably through lactic acid production. However, their low tolerance to conventional sanitizing agents like sulfites limits their broader application. In this context, ozone has emerged as a promising alternative for grape sanitation, offering strong antimicrobial activity while degrading naturally into oxygen, leaving no harmful residues. Compared to other physical methods such as ultraviolet light and pulsed light, ozone presents several advantages, including greater penetration capacity and more uniform action on irregular grape surfaces. These characteristics contribute to a more effective reduction in native microbiota, creating a favorable environment for the implantation of selected non-Saccharomyces yeasts. Ozone treatments, both in gaseous form and dissolved in water, not only reduce indigenous yeast populations efficiently but also promote the metabolic activity of L. thermotolerans during fermentation, as evidenced by increased acidification. These findings highlight ozone as a sustainable and efficient sanitization technique that facilitates the use of unconventional yeasts in winemaking, offering an alternative to sulfites and expanding the possibilities for aromatic and compositional modulation of wine.