We evaluated the occurrence of three antibiotics (roxithromycin, trimethoprim, and chloramphenicol) in surface water and effluents from several sewage treatment plants (STPs) on the Han River, Korea. In addition, standard acute toxicity tests were conducted using the microbe Vibrio fischeri, freshwater macroinvertebrates Daphnia magna and Moina macrocopa, and fish (Oryzias latipes) for these antibiotics. Antibiotics were more frequently detected at higher amounts in effluent samples and in samples collected during the low-flow season. For trimethoprim and chloramphenicol, the levels observed in surface water during low flow were, on average, 108 and 31 ng/L, respectively. These levels were comparable to those measured in the municipal effluents (average, 80 and 37 ng/L, respectively), suggesting the presence of other sources upstream (e.g., livestock wastes). For roxithromycin, surface water levels were approximately an order of magnitude lower than effluent levels. Adverse effects of roxithromycin, trimethoprim, and chloramphenicol were observed at mg/L levels in standard acute aquatic ecotoxicity tests. Based on hazard quotients calculated for the three antibiotics, minimal risks to aquatic systems are suggested. To further increase scientific understanding about the potential impacts of these pharmaceuticals in the environment, however, chronic ecotoxicology studies, with more subtle but ecologically meaningful end points or in combination with other mechanistically related contaminants, may be beneficial.