We studied the effects of four non-nutrient environmental factors (temperature, salinity, irradiance and pH) on the growth inhibition of the macroalgae Ulva pertusa (Chlorophyta) upon the microalgae Heterosigma akashiwo (Rhaphidophyta). Experiments were conducted in single-factor incubation and various two-factor combination experiments in which temperature (10, 15, 25 and 30°C), salinity (10, 20, 30 and 40 g kg−1 water), irradiance (20, 100, 200 and 400 μmol m−2 s−1), and pH (5.5, 7, 8.5 and 10) were varied systematically. The growth rates of U. pertusa and H. akashiwo and the rate of microalgal growth inhibition were altered significantly by changing some of the non-nutrient factors in both the single-factor and the two-factor experiments. The optimal growth conditions for U. pertusa were 20–25°C, salinity of 30 g kg−1, irradiance level of 200–400 μmol m−2 s−1, and pH 8.5–10; optimal conditions for H. akashiwo growth were 25°C, 30 g kg−1, 100 μmol m−2 s−1 and pH 8.5, respectively. The growth inhibitory influence of U. pertusa on H. akashiwo was strongest at 25°C with low salinity (10 g kg−1), high irradiance (400 μmol m−2 s−1) and high alkalinity (pH = 10). The results of this study may be helpful in the development of methods for using green macroalgae to control the proliferation of microalgae in harmful algal blooms (HABs). In particular, these findings provide guidance regarding optimum levels of non-nutrient environmental factors in confined areas, such as aquaculture factories.