Over a period of 5 weeks an experiment was performed with 192 one-day-old Cobb 500 cockerels, which were randomly divided into two experimental groups of 12 cages (replicates) of 8 birds each. The objective of the experiment was to estimate whether the supplementation of a barley-based diet with β-glucanase influences the performance and gastrointestinal ecosystems of broiler chickens in terms of ileal viscosity and pH as well as the concentration of short-chain fatty acids (SCFA) and lactic acid in different segments of the gastrointestinal tract. In the period from day 15 to day 35, dietary enzyme supplementation significantly improved FCR values (1.90 vs 1.85). Body weight gains, liver and caeca weights were not affected by β-glucanase inclusion. Enzyme supplementation decreased ileal viscosity (P≤0.05) and affected SCFA concentrations in the crop and caeca. In digesta from different gastrointestinal segments, lactic acid was detected at the highest levels in the ileum, followed by the crop, gizzard and caeca. The total concentration of SCFAs was found to be highest in the caeca, followed by the ileum, crop, and gizzard. In the crop, the concentrations of acetate, and in the caeca, those of propionate as well as of total SCFA, increased following dietary enzyme supplementation. In none of the examined gastrointestinal segments was the pH value affected (P≤0.05) by enzyme supplementation.