The current study was carried out to explore the effects of lactofeed (LA) as a multi-strain probiotic and succinic acid (SA) on growth variables, gut lactic acid bacteria count, digestive enzymes, innate immune responses, antioxidant capacity, and resistance against Yersinia ruckeri in rainbow trout juvenile (Oncorhynchus mykiss). Three hundred sixty healthy rainbow trout juveniles (13.21 ± 0.41 g) were randomly divided into twelve tanks (300 L) as four experimental groups. They were fed with basal diet (Control; BD), FT1: BD + 1 g/kg LA, FT2: BD + 5 g/kg SA, and FT3: BD + 1 g/kg LA + 5 g/kg SA for eight weeks. According to the results, final weight (FW), weight gain (WG), protein efficiency rate (PER), and feed conversion rate (FCR) revealed a remarkable discrepancy compared to the control group. In addition, dietary inclusion of LA (FT1 and FT3) significantly increased the specific growth rate (SGR). Dietary supplementation of LA+SA (FT3) remarkably improved total bacteria count (TBC) and amylase activity compared to the un-supplemented group. Gut lactic acid bacteria (LAB) count and digestive protease activity in all supplemented fish were remarkably higher than in un-supplemented gruop. Blood immunological factors including white blood cell (WBC) count, total immunoglobulin content (Ig), and the activity of lysozyme (LYZ), alternative complement pathway (ACH50), and acid phosphatase (ACP) were significantly enhanced in the supplemented groups. Neutrophil (NEU) count, skin mucus Ig and hepatic glutathione peroxidase (GPX) increased in SA (FT2 and FT3) treatments. In addition, monocyte (MON) count and skin mucus LYZ activity significantly elevated following feeding with the FT3 diet. Other immunological parameters of skin mucus including protease activity, alkaline phosphatase (ALP) activity, and ACH50 activity, as well as hepatic superoxide dismutase (SOD) and catalase (CAT) activities increased in fish fed with diets containing LA and/or SA. malondialdehyde (MDA) value was remarkably decreased in all supplemented rainbow trout compared to the fish fed with BD. Disease resistance against Y. ruckeri in fish fed with supplemented diets significantly improved with respect to the results obtained in the control specimens. Overall, dietary LA+SA supplementation was beneficial to improve growth performance, gut LAB count, digestive enzyme activities, innate immune responses, antioxidant capacity, and disease resistance in rainbow trout. According to these findings, 1 g/kg LA + 5 g/kg SA is suggested for adding to rainbow trout diet.