Abstract: To investigate the toxic effects of nano-formulations on non-target organisms, this study selected abamectin benzoate and spinosad to compare the acute toxicity and risk of their active ingredients, nano-formulations, and conventional formulations toward Apis mellifera and Eisenia foetida. The results showed: first, the toxicity factor (TF), representing the toxicity change of a formulation relative to active ingredient, was used as an indicator. The TF values for both nano-formulations were ≤4.2, whereas the conventional formulation had the highest TF value of 9.0. This indicates that the toxicity fluctuations of the two nano-formulations did not exceed those of the conventional controls. Second, the acute toxicity of the nanoformulation of abamectin benzoate type II to E. foetida (LC₅₀ = 36.4 mg a.i./kg (soil)) was approximately 1/15 that of the reference emulsifiable concentrate (LC₅₀ = 2.4 mg a.i./kg (soil)). For spinosad type I nanoformulation, the acute oral toxicity toward A. mellifera (LD₅₀ = 0.124 µg a.i./bee) was 1.9 times greater than that of the control suspension concentrate (LD₅₀ = 0.234 µg a.i./bee), whereas its contact toxicity (LD₅₀ = 0.019 µg a.i./bee) was only about one-sixth of the control (LD₅₀ = 0.003 µg a.i./bee). These differences may be attributed to formulation composition, physicochemical properties, test organism species, and exposure routes (oral or contact). The environmental risk of nanopesticides to non-target organisms is a complex function of the inherent toxicity of their active ingredients, formulation components, nano-characteristics, stability, environmental behavior, and exposure scenarios. Therefore, a "case-by-case" approach is recommended for the environmental safety assessments of nanopesticides. Future research should focus on data accumulation and pattern identification to develop rational and targeted methods for the risk assessment of nanopesticides.