Soil microbial communities play a crucial role in vineyard health by supporting nutrient cycling, plant growth, and disease resistance. However, intensive pesticide use can disrupt this balance. Grapevine (Vitis vinifera L.) is particularly vulnerable to Plasmopara viticola, the causal agent of downy mildew, which is commonly controlled with copper-based fungicides. Despite their effectiveness, these treatments pose environmental and health risks. The European Union is promoting sustainable alternatives and aims to reduce pesticide use by 50% in2030. In this context, we evaluated the impact of a new fungicide for downy mildew control on vineyard soil microbiota using a metagenomic approach. A field trial was conducted in an experimental vineyard of the University of Trás-os-Montes e Alto Douro (Portugal) with the cultivar "Tinta Roriz". Sprayings were carried out in 2024 in leaves unfolded until veraison in a total of nine foliar applications. Six different treatments for downy mildew control were tested: M1—control (without any spray); M2 and M3—two concentrations of the new fungicide; M4—new fungicide combined with elicitor (Prevatect® and chitosan-based); M5—new fungicide combined with elicitor (Equiset®, Equisetum arvense L. -based); and M6—conventional fungicide. Soil samples were collected before the first spraying and 15 days after the last round of spraying. Metagenomic libraries for ITS and 16S were prepared and sequenced using the Illumina platform. Proteobacteria and Ascomycota were the most abundant phyla, and Gaiella occulta and Penicillago nodositata the dominant species among bacteria and fungi, respectively. After the sprayings, an increase in bacterial abundance and diversity in the treatments combining the new fungicide with elicitors (M4 and M5) was noted. Fungal species diversity increased in all treatments, except for M1 and M2. Overall, the new fungicide enhanced bacterial abundance and induced changes in fungal diversity, suggesting its potential to positively control downy mildew and disrupt the soil microbial community.