Background Toys, while integral to children’s development and well-being, can serve as significant vectors of infection, especially in hospital settings. This study aimed to assess the effectiveness of toy hygiene education for mothers of hospitalized children on toy contamination and maternal knowledge. Methods A randomized controlled trial was conducted in a public hospital’s pediatric units in Türkiye with 48 mothers and their 1–6-year-old children’s favorite toys. The intervention group (n = 24) received two structured toy hygiene education sessions during hospitalization. Toy surface contamination was measured using an ATP device via swab samples collected from the same toy area on days 1 and 3. Data collection tools included the Personal Information Form, Toy Hygiene Information Form (THIF), and Toy Surface Hygiene Level Record Form (TSHLRF). ClinicalTrials.gov registration: NCT06199804. Results The intervention group demonstrated a significantly higher post-test mean THIF score compared to the control group (F = 227.77, P < .001, η² = 0.835). In addition, toys in the intervention group showed significantly lower ATP contamination levels on day 3 (F = 4.093, P = .048, η² = 0.098). Conclusions Toy hygiene education for mothers significantly improved their knowledge and effectively reduced toy contamination in pediatric hospital settings. These findings highlight the importance of parent-focused hygiene interventions in infection prevention efforts.