Karenia mikimotoi is a common species of red tide dinoflagellate that causes the mass mortality of marine fauna in coastal waters of Republic of Korea. Despite continuous studies on the ecophysiology and toxicity of K. mikimotoi, the underlying molecular mechanisms remain poorly understood. Red sea bream, Pagrus major, is a high-value aquaculture fish species, and the coastal aquaculture industry of red sea bream has been increasingly affected by red tides. To investigate the potential oxidative effects of K. mikimotoi on P. major and the molecular mechanisms involved, we exposed the fish to varying concentrations of K. mikimotoi and evaluated its toxicity. Our results showed that exposure to K. mikimotoi led to an accumulation of reactive oxygen species (ROS) and oxidative DNA damage in the gill tissue of P. major. Furthermore, we found that K. mikimotoi induced the activation of antioxidant enzymes, such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase, in the gill tissue of P. major, with a significant increase in activity at concentrations above 5000 cells/mL. However, the activity of glutathione S-transferase did not significantly increase at the equivalent concentration. Our study confirms that oxidative stress and DNA damage is induced by acute exposure to K. mikimotoi, as it produces ROS and hypoxic conditions in P. major. In addition, it was confirmed that gill and blood samples can be used as biomarkers to detect the degree of oxidative stress in fish. These findings have important implications for the aquaculture of red sea bream, particularly in the face of red tide disasters.