In recent years, there have been a number of deaths caused by the synergistic effects of aminoglycoside (AG) antibiotics in combination with chloramphenicol (CHL). Therefore, combined toxicities of four AG antibiotics: spectinomycin hydrochloride (SPC), succinomycin sulfate (MCR), amikacin sulfate (AMK), tobramycin (TOB) and CHL towards a freshwater organism Vibrio qinghaiensis sp.-Q67 (Q67) were investigated by the time-dependent toxicity microplate analysis method. Thereon, a direct equipartition ray design method was used to design binary mixtures with different concentration ratios. Concentration addition (CA) model was used to analyze toxicity interaction within mixtures. The results showed that: when the exposure time is 12 h, the negative logarithmic pEC₅₀ value of EC₅₀ (median effect concentration) being as toxicity index, the toxicity order of the five antibiotics is TOB>CHL>MCR>AMK>SPC. The combined toxicity characteristics of binary mixture of four AG antibiotics and CHL vary with the composites of the mixture. Generally, most of the cells of Q67 do not change significantly after exposure for 12 h to EC₅₀ of binary mixture, but the damage degree of cells is different after exposure for 12 h to EC₅₀ of single antibiotics. The content of luminescence-related substances in most of the Q67 treated by the mixture with EC₅₀ was lower than that in the control. Combined with the results of SEM (scanning electron microscopy), it was concluded that the mechanism of antibiotics and their mixtures may be through the interference of protein synthesis in luminescent bacteria, leading to bacterial metabolic disorders and even death.