We here employed a model animal of Caenorhabditis elegans to perform toxicity assessment of original surface water samples collected from Three Gorges Reservoir (TGR) in the quiet season in Wanzhou, Chongqing. Using some sublethal endpoints, including lifespan, body length, locomotion behavior, brood size, and intestinal reactive oxygen species (ROS) induction, we found that the examined five original surface water samples could not cause toxicity on wild-type nematodes. Nevertheless, the surface water sample collected from backwater area induced the significant increase in expressions of genes (sod-2 and sod-3) encoding Mn-SODs in wild-type nematodes. Among the examined five original surface water samples, exposure to the original surface water sample collected from backwater area could further cause the toxicity in decreasing locomotion behavior and in inducing intestinal ROS production in sod-3 mutant nematodes. Moreover, the solid phase of surface water sample collected from backwater area might mainly contribute to the observed toxicity in sod-3 mutant nematodes. Our results are helpful for understanding the potential effects of surface water in the TGR region in the quiet season on environmental organisms.