The main intent of the present study was to identify the novel anti-quorum sensing (anti-QS) compounds from probiotic product (Rhodomax) and assess their anti-infection effect of against Vibrio campbellii infection using tomato clownfish (Amphiprion frenatus). In this study, rhodomax probiotic-product was investigated for its active compounds’ prediction through in silico analysis. Initially, anti-QS activity of probiotic-supernatant was authenticated by decrease in bioluminescence production of V. campbellii. As expected, the probiotic-supernatant effectively inhibited the biofilm formation, swimming motility, and hydrophobicity of V. campbellii at 20 μg/ml concentration. During in vivo trail with tomato clownfish, probiotic-supernatant (20 ppm) treatment showed a significant increase in the survival rates to the level of 88% against V. campbellii infection. Further, the reduced colonization of V. campbellii inside the probiotic-supernatant treated animals correlates the anti-infection activity. The histopathological differences in tissue levels such as the gill, intestine, and kidney of infected and probiotic-supernatant treated animals were also support the in-vivo trial, which strongly revealed the disease protection of rhodomax-supernatant against V. campbellii infection. Notably, the docking analysis revealed that the anti-QS mediated anti-infection activity of rhodomax-supernatant against V. campbellii infection is accomplished by the presence of active metabolites such as,4-Dihydro-7-methoxy-2,2-dimethyl-2H-1-benzopyran-3,4-diol and Pyrrolo(1,2-a)pyrazine-1,4-dione, hexahydro-3-(1-methylethyl). Hence, we suggest that these two active metabolites could be the novel alternative agents to control V. campbellii-associated infections in aquaculture.