This study evaluated the synergistic effects of dietary Bacillus subtilis WB60 and mannanoligosaccharide (MOS) in juvenile Japanese eel, Anguilla japonica. Seven treatment diets were formulated to contain three different levels of B. subtilis (0.0, 0.5, and 1.0 × 107 CFU/g diet denoted as BS0, BS0.5, and BS1, respectively) with two MOS levels (0 and 5 g/kg diet denoted as M0 and M5, respectively), and one diet with oxytetracycline (OTC) at 5 g/kg diet. Each diet (BS0M0 (CON), BS0M5, BS0.5M0, BS0.5M5, BS1M0, BS1M5, and OTC) was fed to triplicate groups of 20 fish averaging 9.00 ± 0.11 g (mean ± SD) for eight weeks. Average weight gain, feed efficiency, specific growth rate and protein efficiency ratio of fish fed the BS0.5M5 and BS1M5 diets were significantly higher than those of fish fed CON, BS0.5M0 and OTC diets (P < 0.05). Significant increases in the nonspecific enzymatic activities (e.g., lysozyme and myeloperoxidase) were detected from fish fed the BS0.5M5, BS1M5, and OTC diets compared to the CON, BS0.5M0, and BS0M5 diets (P < 0.05). Whereas, immunoglobulin M expressions were recorded significantly higher for fish fed the BS0.5M5 and BS1M5 diets compared to those of fish fed the other diets (P < 0.05). Also, heat shock protein 70 mRNA levels of fish fed BS0.5M5 and BS1M5 diets were significantly higher than those of fish fed the CON diet (P < 0.05). Histological observations of the intestinal morphology showed healthier gut for fish fed BS0.5M5 and BS1M5 diets than those fed CON, BS0M5, and OTC diets. Additionally, resistance to bacterial challenge with Vibrio anguillarum was recorded significantly lower for fish fed the CON diet than those fed other diets (P > 0.05). Therefore, the results for growth performance, non-specific immune responses, intestinal morphology, and disease resistance demonstrated that supplementation of B. subtilis at 0.5 × 107 CFU/g diet and mannanoligosaccharide at 5 g/kg diet could have beneficial synergistic effects in Japanese eel. The isolated probiotic from eel and the selected prebiotic could lead to the development of a specific and potential synbiotic in Japanese eel aquaculture.