In recent years, peracetic acid (PAA) has gained a lot of attention as an alternative disinfectant to chlorine-based disinfectants in the water industry. Commercial PAA solutions contain both PAA and hydrogen peroxide (HP), and the degradation of HP is slower than PAA when it is used for disinfection. All previous toxicity studies have been based on commercial PAA, and variance in toxicity values have been observed due to different PAA:HP ratios. In this study, the ecotoxicity of pure PAA was studied, eliminating HP from the commercial PAA mixture using potassium permanganate. Ecotoxicity data were obtained by conducting a battery of ecotoxicity tests: bioassays using Vibrio fischeri (V. fischeri), Daphnia magna (D. magna), and Pseudokirchneriella subcapitata (P. subcapitata). The effect concentration (EC50) of pure PAA was 0.84 (a 95% confidence interval of 0.78–0.91) mg/L for V. fischeri and 2.46 (2.35–2.58) mg/L for P. subcapitata, whereas the lethal concentration (LC50) was 0.74 (0.55–0.91) mg/L for D. magna. Compared to this, our previous study found that the EC50 values of commercial PAA towards V. fischeri and P. subcapitata were 0.42 (0.41–0.44) and 1.38 (0.96–1.99) mg/L, respectively, which were lower than pure PAA, whilst the LC50 for D. magna was 0.78 (0.58–0.95) mg/L. These results showed that pure PAA was less toxic to the most commonly used aquatic species for toxicity tests compared to commercial PAA, except for D. magna.