Nonylphenol (NP) is a contaminant that has negative impacts on aquatic organisms. To investigate its phytotoxicity, bioaccumulation, and degradation in algae without associated bacteria, six freshwater microalgae—Ankistrodesmus acicularis, Chlorella vulgaris, Chroococcus minutus,, Scenedesmus obliquus, Scenedesmus quadricauda, and Selenastrum bibraianum—in bacteria-free cultures were studied. When exposed to 0.5–3.0 mg L⁻¹ NP for 4 days, cell growth and photosynthesis, including maximal photochemistry (Fv/Fm), were suppressed progressively. The antioxidant responses of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) showed species differences. While the antioxidant enzymes in C. vulgaris and S. obliquus were more active with the increase of NP (0–3 mg L⁻¹), they dropped in the other four algae at concentrations of 1 and 1.5 mg L⁻¹. Therefore, C. vulgaris and S. obliquus were designated as NP-tolerant species and showed more conspicuous and faster changes of antioxidant reactions compared with the four NP-sensitive species. All six species degraded NP, but A. acicularis was more reactive at low NP concentrations (<1 mg L⁻¹), suggesting its possible application in sewage treatment for its potential for effective NP removal from water bodies in a suitable scope. Therefore, the conclusion is that biodegradation of NP by algae is species specific.