To understand how environmental conditions affect the allelopathic inhibition of toxic Microcystis aeruginosa by berberine, the independent effects of some environmental factors, including temperature, light, and aeration, on the growth and extracellular microcystin (MC) content of M. aeruginosa (FACHB 905) treated with 0.000 and 0.001% (w/v) berberine were investigated. The results showed that higher temperature and light density, and aeration in daytime were beneficial for the growth of M. aeruginosa under the measured environmental conditions. The allelopathic effects of berberine on M. aeruginosa were closely associated with the environmental conditions. Berberine had the best inhibitory effects when temperature, light and aeration were more optimal for growth. In darkness, no changes in the density of M. aeruginosa were observed with the prolongation of culture time and berberine could hardly exhibit algicidal effects. Disturbance in the photosynthesis process might be one of the main reasons responsible for algicidal function. Berberine could increase extracellular MC contents significantly via killing and lyzing algal cells. Other treatments coupled with berberine needed to be carried out to degrade or remove MC released from berberine-killed algal cells.