Pelargonic acid is a naturally occurring fatty acid that has the advantages of low biotoxicity and a short half-life. Pelargonic acid is used as non-selective herbicide in agriculture. It can penetrate the wax cuticle of plants and generate peroxide under light, rapidly causing the burn-down effect in weeds. The domestic evaluation data regarding the influence of pelargonic acid on aquatic environments is currently insufficient. This study sought to establish the median lethal concentrations of a pelargonic acid technical material (98.5%) and emulsion (80%) containing surfactant for the species Pseudorasbora parva, Oreochromis niloticus, Anguilla japonica, Neocaridina denticulate, Macrobrachium rosenbergii and Sinotaia quadrata. The 96-hour median lethal concentrations of the pelargonic acid technical material for those species were 121.38, 238.93, 267.11, 313.84, 417.29 and 159.65 mg/L, respectively, while the 96-hour median lethal concentrations of the pelargonic acid emulsion for those species were 15.94, 31.14, 18.24, 242.84, 178.90 and 51.81 mg/L, respectively. The toxicity of the pelargonic acid emulsion was higher than that of the technical material, and the shrimps had the highest tolerance of both the technical material and the emulsion. After O. niloticus was immersed in the pelargonic acid technical material and the emulsion at the median lethal concentrations, an irregular opercular beat frequency and hemorrhaging in the gill filaments were observed. The results indicated that pelargonic acid is irritating to fish gills, and confirmed that pelargonic acid impairs the oxygen uptake of gills and leads to asphyxia mortality.