Infectious disease outbreaks are one of the major challenges in aquaculture; therefore, there is an increasing interest in the application of probiotics for sustainable aquaculture practices to minimize the spread of infectious diseases. In this study, bacteria were isolated from Pinctada radiata to examine their probiotic potential. Lactic acid bacteria (LAB) are potentially good probiotic candidates for aquaculture. Five LAB strains were isolated from the gut contents of the pearl oyster P. radiata (Leach, 1814) on the Jeddah coast of the Red Sea. The LAB were characterized morphologically, biologically, and biochemically, examined for their probiotic properties, and identified using 16S rRNA sequencing. The five selected isolated LAB strains were Gram positive (rods and cocci) and tested negative for catalase and oxidase. The LAB strains were identified as Bacillus valezensis (POR1), B. siamensis (POR2), Staphylococcus epidermidis #1 and S. epidermidis #2 (POR3 and POR5), and S. hominis (POR4). POR1 and POR2 tested negative for gamma-hemolytic activity, while POR3, POR4, and POR5 tested positive for alpha-hemolytic activity. All five strains showed sensitivity to antibiotics, including erythromycin (E10), nitrofuration (F100), and novobiocin (NV5), and four strains showed an acid tolerance up to  2.5 pH . Among the isolates, POR1, 2, 4, 5 tolerated 3-h exposure to 0.3% bile salt. All LAB strains showed an antagonistic activity against clinical strains of S. aureus, Acinetobacter baumanii, methicillin-resistant S. aureus, and Escherichia coli. Therefore, it could be suggested that the five LAB strains isolated from the gut contents of P. radiata could be good candidate probiotics for aquaculture application since those strains showed significance of probiotic selection tests.