Experiments were carried out to investigate the effects of nutrients, salinity, pH and light:dark cycle on growth rate and production of reactive oxygen species (ROS) by Chattonella marina, a harmful algal bloom (HAB) species that often causes fish kills. Different nitrogen forms (organic-N and inorganic-N), N:P ratios, light:dark cycles and salinity significantly influenced algal growth, but not ROS production. However, iron concentration and pH significantly affected both growth and ROS production in C. marina. KCN (an inhibitor of mitochondrial respiration) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (an inhibitor of photosynthesis) had no significant effects on ROS production. Vitamin K3 (a plasma membrane electron shuttle) enhanced ROS production while its antagonist, dicumarol, decreased ROS production. Taken together, our results suggest that ROS production by C. marina is related to a plasma membrane enzyme system regulated by iron availability but is independent of growth, photosynthesis, availability of macronutrients, salinity and irradiance.