The two-spotted spider mite Tetranychus urticae Koch (Acari: Tetranychidae) is a major pest of horticultural, ornamental, fruit, and strawberry crops worldwide. Currently, various management tools have been explored for this pest, with nanoparticles being one of them, which stand out for their characteristics and multiple effects. This study evaluated the effects of green-synthesized gold nanoparticles (AuNPs) on the mortality and repellency of T. urticae and its natural predator Phytoseiulus persimilis under laboratory conditions, as well as their efficacy in greenhouse tomatoes against T. urticae. In the laboratory, a biological window for AuNPs (50–100 mg L⁻¹) on the pest and predator was established using a residual film method and a free-choice assay. In the greenhouse, four concentrations (300, 500, 750, and 1000 mg L⁻¹) were evaluated via foliar application at 10-day intervals. The results showed susceptibility to AuNPs in all stages of T. urticae and the adult P. persimilis. The death times from AuNPs were similar in both species. Furthermore, the AuNPs were selective for the pest rather than the natural enemy. In greenhouses, AuNPs affected T. urticae populations in tomato plants, and significant differences were observed on some continuous and final agronomic variables (associated with fruits). This study showed that T. urticae and P. persimilis were susceptible to green-synthesized AuNPs. AuNPs can be a management tool, although studies on other non-target species and estimating agronomic effects on other crops are recommended.