Steroid estrogens (SEs) play a significant role as endocrine-disrupting substances, and one of their major sources is animal manure. However, there is limited information available regarding the loss of SEs in farmland soil after the application of commercial composted animal manure or fertilizers. To address this gap, our study aimed to simulate rainfall and flood irrigation scenarios and investigate the loss characteristics of SEs, as well as Chemical Oxygen Demand (COD), Total Nitrogen (TN), and Total Phosphorus (TP) in runoff from soil–manure mixtures. The results demonstrated that the loss concentrations of SEs (73.1 ng/L of the mean E2β active equivalent factor) presented a potential environmental risk. Additionally, substituting composted manure with commercial organic fertilizers lead to a significant reduction in TP (maximum 56%) and TN (maximum 24%) loss. Consequently, the application of commercial organic fertilizers offers considerable advantages in maintaining nitrogen and phosphorus fertilization efficiency while controlling SEs loss. Furthermore, our study explored the synergistic pollution mechanism among these pollutants and observed significant correlations between SEs and TN, TP, and COD loss concentrations, indicating the simultaneous occurrence and migration of these pollutants in agricultural non-point source pollution. These results provide valuable insights into the environmental risk associated with SEs from agricultural non-point sources.