Vibriosis, a collective Vibrio infection, is a widespread epizootic disease that affects most wild and farmed fish species worldwide, and is currently the major limiting factor in the development of intensive mariculture industry. In our previous study, we found that a Bacillus pumilus strain, D5, isolated from white shrimp (Litopenaeus vannamei) exhibited antibacterial ability against Vibrio alginolyticus, V. harveyi and V. parahaemolyticus. In order to get mutants with higher antibacterial abilities, the B. pumilus D5 strain was irradiated with ultraviolet (uv) radiation and treated with nitrosoguanidine (NTG). After three screenings, four mutant strains named B. pumilus mutant NG24, NG25, UV35 and UV37 were selected. The experimental results showed that the average bacteria inhibition zones against V. harveyi and V. parahaemolyticus of these four mutant strains provide significantly higher inhibition than B. pumilus D5 (p<0.05). In a co-culture test, the B. pumilus D5 strain and its mutants suppressed the growth of V. parahaemolyticus in vitro only if the initial inoculum of the given strain was up to 100000 times higher than the inoculum of V. parahaemolyticus. Furthermore, B. pumilus NG25 showed higher antagonistic abilities against pathogenic V. harveyi and V. parahaemolyticus than B. pumilus D5 (p<0.05). These study results reveal that the B. pumilus D5 mutants have potential applications for controlling pathogenic Vibrio spp. in aquaculture practices.