There is an urgent need to develop microbial control strategies to control vibriosis outbreaks in aquaculture industries. The present work was devoted to screening for aquatic pathogenic Vibrio spp. antagonistic bacteria in marine fishes and shrimp. The sensitivities of the probiotic to various antibiotics were tested as well. In the present study, a total of 84 isolates were isolated from four samples. All the isolates were tested against pathogenic Vibrio spp., including V. alginolyticus, V. parahaemolyticus, and V. harveyi. Among the 84 isolates, the strain D5, isolated from white shrimp, showed high antagonistic abilities toward pathogenic Vibrio spp. The strain D5 was identified as Bacillus pumilus by Gram stain and 16S rRNA sequences, as well as by its physiological and biochemical characteristics, and was thus named Bacillus pumilus D5. The B. pumilus D5 exhibited antagonism against V. alginolyticus, V. anguillarum, V. cholerae, V. harveyi, V. parahaemolyticus, and V. vulnificus. Moreover, Escherichia (E.) coli and Salmonella typhimurium were also inhibited by the B. pumilus D5. To test the sensitivity of the B. pumilus D5, 15 different antibiotics were used. The B. pumilus D5 exhibited at least some sensitivity to 14 different antibiotics tested in this study, while it was resistant to lincomycin. In conclusion, the B. pumilus D5 exhibited antagonism against V. alginolyticus, V. anguillarum, V. cholera, V. harveyi, V. parahaemolyticus, and V. vulnificus, as well as sensitivity to most of the antibiotics tested in this study. The present study results reveal that the B. pumilus D5 has potential applications for controlling pathogenic Vibrio spp. in aquaculture practices.