Nonylphenol ethoxylates (NPEOs) are widely utilized in pesticide formulations and industrial products but are known for their endocrine-disrupting properties. Consequently, substitutes such as tristyrylphenol ethoxylates (TSPEOs) have been introduced as inert ingredients in pesticide formulations. Here, we showed that TSPEOs exhibited subacute toxicity in male mice. For the first time, we studied the differences in subacute toxicity (28-day exposure) and the potential toxic effects of TSPEOs with varying polymerization degrees, specifically agricultural emulsifier (AE) #602 and AE #604, in male mice. We demonstrate that AE #602 can induce liver injury, as evidenced by hepatocyte swelling and vacuolar degeneration across all treated groups, along with significant hepatocellular necrosis in the high-dose group. These pathological changes were associated with alterations in oxidative stress biomarkers, including a significant decrease in malondialdehyde levels (0.57 times in the high-dose group, p < 0.05) and increased activities of glutathione peroxidase (up to 1.27 times, p < 0.05) and glutathione, suggesting a potential adaptive compensatory response. Both TSPEOs were found to cause gastric injury according to the results of organ indices and histopathological analyses. AE #604, with lower polymerization degree, caused more severe gastric injury than AE #602. Our findings indicate that NPEO substitutes should be tested for hepatotoxicity and gastrotoxicity and highlight the need for further research into the toxicity differences induced by varying degrees of polymerization of TSPEOs on human health.