Due to the discharge of metabolic waste in feces and the accumulation of uneaten feed, cultural ponds are easily polluted by massive ammonia nitrogen, nitrite, and organic matter. It deteriorates the cultivation environment and usually causes prevalence of diseases in fish. Therefore, the improvement of the cultivation environment and the reduction of infection opportunity for pathogens are the key factors for enhancing survival rate. The aim of this research is to investigate the effects of the probiotic, photosynthetic bacteria Rhodovulum sulfidophilum and Bacillus subtilis on the inhibition of several Vibrio pathogens in orange-spotted grouper, Epinephelus coioides aquaculture. In vitro, the R. sulfidophilum and B. subtilis at the concentration of 104 CFU/ml effectively inhibited the multiplication of Vibrio alginolyticus, V. anguillarum, and V. parahaemolyticus in plates. The addition of R. sulfidophilum and B. subtili into water can mitigate the amounts of ammonia nitrogen and nitrite in the ponds and thus stabilize water quality. The research showed that adding R. sulfidophilum and B. subtilis to the grouper culture pound can stabilize water and inhibit pathogens; create a good nursery environment for grouper culture ; reduce the prevalence of disease; and elevate the breeding gains.