This study aimed to investigate in vitro the probiotic potential of three yeasts strains (BB06, OBT05, and MT07) isolated from agro-food natural sources. Screening was performed, including several functional, technological, and safety aspects of the yeast strains, in comparison to a reference Saccharomyces boulardii, to identify the ones with suitable probiotic attributes in aquaculture. The yeast strains were identified by 5.8S rDNA-ITS region sequencing as Metschnikowia pulcherrima OBT05, Saccharomyces cerevisiae BB06, and Torulaspora delbrueckii MT07. All yeast strains were tolerant to different temperatures, sodium chloride concentrations, and wide pH ranges. S. cerevisiae BB06 showed a strong and broad antagonistic activity. Moreover, the S. cerevisiae strain exhibited a high auto-aggregation ability (92.08 ± 1.49%) and good surface hydrophobicity to hexane as a solvent (53.43%). All of the yeast strains have excellent antioxidant properties (>55%). The high survival rate in the gastrointestinal tract (GIT) can promote yeast isolates as probiotics. All yeast strains presented a resistance pattern to the antibacterial antibiotics. Non-hemolytic activity was detected. Furthermore, freeze-drying with cryoprotective agents maintained a high survival rate of yeast strains, in the range of 74.95–97.85%. According to the results obtained, the S. cerevisiae BB06 strain was found to have valuable probiotic traits.