Background <p>While hand hygiene is a primary defense against infection-related symptoms, the specific dose-response relationship and the influence of psychological factors (e.g., depression) on this association in children are not well understood.</p> Purpose <p>To examine the dose-response relationship between multi-scenario hand hygiene behaviors and infection-related symptoms and to test the moderating effects of depression in school-aged children in China.</p> Methods <p>This multicenter, cross-sectional study enrolled 22,602 students from 54 schools. Hand hygiene was assessed using a composite score (0–3) across three key scenarios. Infection-related symptoms was defined by the 14-day incidence of seven predefined symptoms (e.g., respiratory, gastrointestinal). Depression was assessed using the Center for Epidemiologic Studies Depression (CES-D) scale. Three-level mixed-effects logistic regression models were employed to examine dose-response relationships, accounting for county- and school-level clustering. To ensure the robustness of our findings, we calculated E-values to quantify potential unmeasured confounding and performed subgroup analyses across infection-related symptoms types and demographics to assess the stability of the association. Additionally, a moderation analysis examined the interaction between hand hygiene and depression, specifically among secondary school students.</p> Results <p>Multilevel analysis revealed a significant nonlinear dose-response relationship, with a threshold at score 3 compared to scores 0–2 (adjusted odds ratio [<i>AOR</i>] = 0.676, 95% confidence interval [<i>CI</i>]: 0.633–0.721, <i>P</i> &lt; 0.001). The association remained robust after subgroup analyses, and E-values ≥ 1.73 indicated resilience to potential unmeasured confounding. In the moderation analysis for secondary-school students, depression significantly moderated the association between hand hygiene and infection-related symptoms (<i>P</i>_{<i>interaction</i>}&lt;0.001). The association remained significant for students without depression (<i>AOR</i> = 0.832, <i>P</i> &lt; 0.001), but was attenuated and non-significant in those with depression (<i>AOR</i> = 1.010, <i>P</i> = 0.802).</p> Conclusions <p>Maintaining hand hygiene across all three scenarios (score = 3) was associated with a lower risk of infection-related symptoms in school-age children. However, the presence of depression may attenuate this association. School-based infection control may need to transition from purely behavioral interventions (e.g., reinforcing hygiene) in primary schools to an integrated approach that includes mental health support in secondary schools.</p> Clinical trial number <p>Not applicable.</p>