The short-spined urchin, Tripneustes gratilla is a fast-growing, shallow water echinoid, occurring throughout the waters of the tropical Indo-Pacific. The species produces large gonads with excellent market acceptance, and is one of the most commercially important sea urchin species in countries, such as Japan. High demand has led to overfishing of natural populations prompting many countries including South Africa to explore the possibility of sea urchin aquaculture. With the increasing intensification and commercialization of aquaculture production, mass mortality during the larval stages usually associated to the presence of opportunistic pathogens is a major problem . With an increasing demand for environment- friendly aquaculture, the use of alternatives such as probiotics is now widely accepted. This study aimed to isolat e and identify potential probiotics from the digestive tract of sea urchin (Tripneustes gratilla ) to increase survival of the urchin larval phase . Nine wild-caught sea urchins were sacrificed for the isolation of potential probiotic bacteria and the digestive tract bacteria grown on selective media . A total of 250 isolates were recovered of which 27 showed antagonistic properties (zones of inhibition) against six known marine pathogens. The strains producing the top ten zones of inhibition were identified using a MALDI-TOF mass spectrometer and the Biotyper software ( Bruker Daltonics MALDI-TOF biotyper RTC 3.0) . Candidate probionts were capable of producing digestive enzymes (protease and amylase) with hydrolyzed skim milk and starch halos ranging from 7–28 mm. These results show that bacteria obtained from the indigenous microbiota of sea urchin could be used as probiotics in aquaculture.