An 8-weeks feeding trial was performed to investigate the possible effects of supplementation of Nile tilapia diet with Cordyceps militaris spent mushroom substrate (SMS) single or combined with Lactobacillus plantarum on immune parameters and growth performance. For this aim, Nile tilapia fingerlings were fed with four experimental diets namely: Diet 1 (0 – control), Diet 2 (10 g kg−1 SMS), Diet 3 (108 CFU g−1L. plantarum), and Diet 4 (10 g kg−1 SMS + 108 CFU g−1L. plantarum). At the end of feeding trial, skin mucus parameters, serum immune parameters, and growth performance were measured. The results indicated that supplementations SMS + L. plantarum or/and resulted in a significant increase in skin mucus lysozyme and peroxidase activities compared with the control group after 8 weeks of feeding trial (P < 0.05). The highest values of these parameters were recorded for fish fed both SMS + L. plantarum supplementations. Nonetheless, no significant difference was recorded between other supplemented groups (P < 0.05). For serum immunology, the results showed that serum lysozyme activity, alternative complement, phagocytosis, serum peroxidase, and respiratory burst activities were significantly higher in supplemented groups compared to the control (P < 0.05). The highest values were recorded in fish fed both SMS and L. plantarum with respect to the individual application. No significant differences were observed between fish fed SMS and L. plantarum (P < 0.05). Results on growth performance indicated that fish fed supplemented diets showed a statistically significant increase in the specific growth rate (SGR), weight gain (WG), final weight (FW) compared to the control group (P < 0.05). The highest SGR and WG values were observed in fish fed both dietary SMS and L. plantarum. However, no significant differences in these parameters were observed in fish fed SMS or L. plantarum alone (P > 0.05). The FCR was significantly lower in fish fed 10 g kg−1 SMS + 108 CFU g−1L. plantarum than in other groups, while control group presented the highest values (P < 0.05). The present results suggested that the combination of these natural substances could be considered as potential feed-additives for aquaculture farmed fish.