To reduce the impact of synthetic insecticides on human health and the environment, eco-friendly alternatives must be investigated. Knowledge of the side effects on pests and natural enemies of natural products applied to vineyards is very useful. Sulfur dust, which is used in vineyards to control powdery mildew, is investigated in laboratory and field bioassays for its effects on Lobesia botrana egg laying, egg hatching, and larval settlement. In field trials, the efficacy of sulfur dust against the two L. botrana carpophagous generations is compared with that of Bacillus thuringiensis and kaolin, and its side effects on the phytoseiid mite Kampimodromus aberrans are evaluated. In the bioassays, sulfur dust reduced female survival by 43%, egg laying by around 80%, egg hatching by 10%, and larval settlement by 55%. In field trials, sulfur dust caused a significant decrease in the number of L. botrana larval nests of both generations, even though the efficacy was lower than that of B. thuringiensis. No negative effects of sulfur dust on the predatory mite population density was observed. On the basis of these results, in the context of Integrated Pest Management strategies in vineyards, the activity of sulfur dust against L. botrana could be exploited by timing its application to the beginning of egg laying.