Cu2O NPs/diatomite is a composite material synthesized by chemical reduction method of Cu2+ ion adsorbed on diatomite. Cu2O NPs/diatomite is capable of adsorption of pollutants and microbial treatment, for aquaculture water treatment applications. The study focused on the acute toxicity determination of Cu2O NPs/diatomite material on Swiss white mice by oral tests. The results showed that Cu2O NPs/diatomite was almost non‐toxic with a test dose of 2,000 mg/kg of mice body weight, LD50 could not be determined. Monitoring the mice's activity after 14 days, the mice have normal activities, normal stool, urine, pink mucosa, smooth feather, good reflexes. Thus, with the very low acute toxicity on white mice, Cu2O NPs/diatomite was evaluated a good candidate for aquaculture treatment application.