Abstract <p>The task of optimizing the quality of feed and, hence, the rate of biomass accumulation by fish in aquaculture requires improved knowledge on the activity of their digestive enzymes and the composition of intestinal microbiota. This is the first study of the activity of glycosidases and proteinases in the intestinal mucosa and chyme, as well as of the intestinal microbiome composition, in the African catfish <i>Clarias gariepinus</i> grown in tanks with a closed water supply system. The total amylolytic and total proteolytic activities in the intestinal chyme were significantly higher than those in the mucosa, and higher in small fish than in large ones. The activity of disaccharidases (sucrase and maltase) in the intestinal mucosa did not depend on the fish size. In both size-age groups of African catfish, the following bacterial phyla were found to be dominant: <i>Pseudomonadota, Bacillota, Actinomycetota, Fusobacteriota</i>, and <i>Bacteroidota</i>. In the group of small fish, <i>Pseudomonadota</i> accounted for almost half of the number of intestinal bacteria, whereas in large catfish, the distribution of these bacteria phyla was more uniform. For the first time, the relationship between the activity of digestive enzymes and the relative abundance of bacteria of various taxa was revealed. The highest positive correlation in the chyme of small catfish was found for the total proteolytic activity with abundance of bacteria of the order <i>Lactobacillales</i> of the class <i>Bacilli</i>. In large catfish, the highest positive correlations were established for the total proteolytic activity of the intestinal chyme and abundance of bacteria of the orders <i>Actinomycetales</i> and <i>Bacillales</i>, and for the total amylolytic activity, with abundance of bacteria of the orders <i>Fusobacteriales</i> and <i>Clostridiales</i> (<i>Eubacteriales</i>). The data obtained can be used when developing nutritional plans for catfish in aquaculture.</p>