Multi-endpoint toxicity tests were used to evaluate the acute toxicity, estrogenic activity, neurotoxicity, genotoxicity, and ecological risks of surface water and sewage from possible pollution sources in rural areas of the Yellow River (China). Toxicity testing results showed that the luminescence inhibition rates of acute toxicity ranged from not detected (ND) to 38%, the 17β-estradiol equivalent (E2-EQ) values of estrogenic activity ranged from 4.8 to 131.0 ng·L−1, neurotoxicity was not detected, and the protein effect level index (PELI) values of genotoxicity ranged from 1 to 6.06. Neither acute toxicity nor genotoxicity were detected in the tributaries of the Yellow River (River 2) flowing through the investigated rural area. The distribution of high estrogenic activity sites was relatively scattered, but mainly located in the tributaries of River 2. Industrial, domestic, and livestock and poultry breeding sewage were all possible sources of toxicity, and the contribution of livestock and poultry to environmental estrogens in the surface water was significant. Furthermore, the potential effect-targeting risks of toxic substances in the surface water for aquatic organisms were assessed using the risk quotient method, by considering the toxic equivalent concentration. The results indicated that the risk of estrogenic activity was the main ecological risk in the surface water of this rural area. Except for the reservoir site, the other sampling sites showed a moderate to high estrogenic activity risk, especially in the tributaries of River 2.