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Compatibility of each strain in a multi-strain probiotic (MSP), along with its properties, becomes a strong base for its formulation. In this study, single-strain probiotics (SSPs) and multi-strain probiotics (MSPs) were evaluated in vitro for strain compatibility, microbial antagonism, biofilm formation capacity, and stress tolerance. <i>Bacillus amyloliquefaciens</i> L11, <i>Enterococcus hirae</i> LAB3, and <i>Lysinibacillus fusiformis</i> SPS11 were chosen as MSP1 candidates because they showed much stronger antagonism to <i>Aeromonas hydrophila</i> and <i>Streptococcus agalactiae</i> than a single probiotic. MSP 2 candidates were <i>Lysinibacillus fusiformis</i> strains SPS11, A1, and <i>Lysinibacillus sphaericus</i> strain NAS32 because the inhibition zone produced by MSP 2 against <i>Vibrio harveyi</i> and <i>Vibrio parahaemolyticus</i> was much higher than that produced by its constituent SSPs. MSP1 in the co-culture assay reduced (<i>p</i> < 0.05) <i>A. hydrophila</i> count from 9.89 ± 0.1 CFU mL⁻¹ to 2.14 ± 0.2 CFU mL⁻¹. The biofilm formation of both MSPs were significantly higher (<i>p</i> < 0.05) than its constituent SSPs and the pathogens. The SSPs in both MSPs generally showed resistance to high temperatures (80, 90, and 100 °C) and a wide range of pH (2 to 9). This in vitro assessment study demonstrates that MSP1 and 2 have the potential to be further explored as multi-strain probiotics on selected aquatic species.