Plasmopara viticola (Pv), the causal agent of downy mildew, is one of the most damaging pathogens affecting grapevine. Current control strategies largely depend on copper-based fungicides and synthetic chemicals, raising increasing concerns related to environmental sustainability and pathogen resistance. This study evaluated the efficacy of a novel Saccharomyces cerevisiae extract (U) as an inducer of resistance in the grapevine–Pv interaction. Microscopic observations revealed the ability of U to inhibit Pv spread over the leaf. Additionally, biochemical and molecular responses were analyzed in grapevine leaves subjected to four treatments: plants treated only with water (U⁻/Pv⁻; i.e., control) or U (U⁺/Pv⁻), inoculated with Pv (U⁻/Pv⁺), or both treated with U and then inoculated with Pv (U⁺/Pv⁺). Fully expanded leaves were sampled at 2-, 5-, 24-, and 72-h post inoculation (hpi). In U⁺/Pv⁻ leaves, jasmonic, salicylic and abscisic acid (JA, SA, and ABA), as well as hydrogen peroxide (H₂O₂) increased at 2 hpi (+44, +33, +38%, and 3-fold, respectively), accompanied by upregulation of pr1 (2-fold higher than control, respectively), suggesting the capacity of U to trigger the plant alert system. In U⁻/Pv⁺ leaves, peaks of JA and H₂O₂ occurred at 24 hpi (+40% and 4-fold higher than control), followed by marked ethylene emissions and upregulation of pr1 and pr2 (i.e., genes associated with Pv defense; around 2-fold, averagely) at 72 hpi, confirming the progression of infection. In contrast, U⁺/Pv⁺ leaves showed stronger peaks of H₂O₂ at both 2 and 5 hpi (7-fold and +58%, respectively), together with SA accumulation and upregulation of pr1, pr2, eds1, and chit1b at 72 hpi (more than 2-fold), suggesting a priming effect of U. Overall, U effectively enhanced grapevine defense responses and limited Pv development, highlighting its potential as a sustainable disease management strategy.