Grapes represent a significant agricultural product globally. Despite their significance and value, grapes face numerous challenges, including disease. Grapevines are susceptible to various pathogens, with downy mildew caused by Plasmopara viticola, being the most damaging. In the twentieth century, contact and systemic fungicides were developed to combat plant pathogens, such as downy mildew. Recently, there has been a growing demand to minimize fungicide use and shift towards sustainability through implementing eco-friendly practices. Utilizing plant defense elicitors to stimulate disease resistance in grapevines is a crucial strategy. We conducted experiments in the field over two seasons, 2021 and 2022, at two different locations using two grape cultivars (‘Flame’ and ‘Crimson’). Four concentrations of chitosan (1, 2, 4, and 8 mM) were applied. The results indicated that chitosan could induce systemic resistance against downy mildew caused by P. viticola by activating the salicylic acid (SA) pathway, increasing the plant’s concentrations of SA and phenols, and augmenting the efficacy and activity of defense enzymes. The treatment also significantly enhanced the yield production of 1.3-glucanase, chitinase, peroxidase, and polyphenol oxidase. The present study assessed the efficacy of chitosan as a resistance inducer in managing grapevine downy mildew and examined its mechanism of action.