Antibiotics, as special pollutants in piggery wastewater, have negative effects on wastewater treatment processes. However, it is difficult to obtain a clear understanding of the toxic potency of antibiotics using the common inhibitory experiments of wastewater because of their low concentrations. Therefore, this study for the first time reports research on examining the toxic strength of piggery wastewater containing antibiotics by acute toxicity experiments using luminescent bacteria. Also reported herein for the first time were the mixed toxic effects of different antibiotics on luminescent bacteria using orthogonal experimental designs. Results showed that piggery wastewater had high ecotoxicity equal to 24.6 mg·L^-1 HgCl₂, and the toxicity units (TU) of chlortetracycline, amoxicillin, florfenicol, and sulfamethazine (SM₂) were 85.5, 64.72, 1.27, and 1.20, respectively, which fell under high or significant ecotoxicity according to the Toxicity Classification System (TCS). The IC₁₀ values indicated that the antibiotics could impose single or mixed toxic effects on luminescent bacteria even in trace concentrations. Among these antibiotics, chlortetracycline was the most significant one needing more attention in the treatment of wastewater-containing antibiotics.