The aim of this research was to evaluate the possible positive effects of feeding Nile tilapia (Oreochromis niloticus) with a diet containing a commercial probiotic AQUA-PHOTO ® [Lactobacillus plantarum (1.51 × 10 6 CFU g −1 ) and Bacillus subtilis (1.34 × 10 7 CFU g −1 )] on digestive enzymes' activity and gut microbiome profile. The experimental design was completely randomized, with two treatments [control and probiotic diets (0.08 g.100 g −1 of feed)] and six replicate tanks per group (n = 8 animals per experimental unit, mean weight 8.18 g ± 1.85 g). The 96 fish were distributed into 12 aquaria and fed with the probiotic and control diets for 7 days. After this period, intestine samples (anterior, middle, and posterior portions) were collected from two individuals per tank. The samples were analyzed for assessment of DGGE profile and digestive enzymes' activity (amylase, alkaline protease, and alkaline phosphatase). The results were subjected to analysis of variance (ANOVA) and the means compared by t test (P < 0.05). The addition of B. subtilis and L. plantarum in the diets modulated the intestinal microbial biodiversity, more specifically the species richness and habitability index (P < 0.05). Besides, probiotic feeding also increased specific amylase activity in the anterior gut (P < 0.05). The other variables studied did not show any significant differences between treatments. The oral administration of B. subtilis and L. plantarum (AQUA-PHOTO ® ) modulates intestinal microbiota profile and increases the amylase enzymatic activity. Therefore, this measure may provide better utilization of carbohydrate sources in the diet, supplying energy for Nile tilapia growth.