Abstract: With the increase of antimicrobial resistances due to the widespread use of antibiotics, the search of new probiotics to control aquaculture diseases has a growing public interest. The aim of this study was to isolate bacteria with antimicrobial effect from the gut of marine healthy fishes and select lactic acid bacteria (LAB) as potential probiotics, being strains considered as generally regarded as safe (GRAS) by the European Food Safety Agency (EFSA). Of a total of 45 Gram-positive strains with antimicrobial activity found in a screening of the gut microbiota of 13 marine fishes, nine were identified as LAB by 16S rRNA gene sequencing. LAB strains (five Lactococcus lactis subsp. lactis, two Enterococcus spp., one Lactobacillus plantarum, and one Leuconostoc mesenteroides subsp. mesenteroides) also showed a broad-spectrum antibacterial activity against aquaculture pathogens such as Vibrio harveyi, V. splendidus, and Photobacterium damselae and survived in experimental gastrointestinal conditions when grown in culture media modified with different values of pH and bile salts. These results showed the potential of LAB obtained from the indigenous microbiota of wild marine fishes for use as probiotics in aquaculture.