As two typical tetracyclines, chlortetracycline (CTC) and oxytetracycline (OTC) coexist widely in water. In the experiment, Myriophyllum aquaticum (Vell.) Verdc was exposed to the orthogonal hydroponic environment with the concentrations of CTC and OTC at 1, 10, and 50 mg/L for 7 days (7 D) and 14 days (14 D). The results showed that the plant height, relative growth rates (RGR), and photosynthetic pigment contents of M. aquaticum decreased significantly after 14 D of CTC/ OTC, which was stronger than that after 7 D, and the toxicity of CTC was stronger than OTC. The combined toxicity of the two on the RGR and total chlorophyll content was mainly synergistic or additive after 7 D. After 14 D, the interaction changed to antagonism or addition, and the response of total chlorophyll content was more obvious than that of RGR. After 7 D of combined treatments, the malondialdehyde (MDA) content decreased significantly, superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities responded to different combined concentrations, respectively, and CAT was the main protective enzyme of M. aquaticum. After 14 D of combined treatments, the hydrogen peroxide (H2O2) content increased significantly, the MDA content increased first and then decreased, and POD was the main protective enzyme among the three antioxidant enzymes. Overall, this study provided the first insight into the single and combined toxicity of CTC and OTC on the growth and physiology of M. aquaticum at different time periods, revealing the reversal of the combined toxicity with time of exposure.