This study aims to define the changes in the quality of bakery products depending on the β-glucan source and its contribution using bake-off technology. The examined bread was enriched with a 10% addition of oat flour, barley flour, oat fibre preparation, and barley fibre preparation. Bread was tested for rheological parameters, baking performance, hardness and springiness, water content, specific volume, porosity, crust and crumb colour, and β-glucan content. In the executed research, the adverse effect of this component on the formation of gluten network and hardness of the crumb was observed. In the double compression test, it was shown that the highest hardness on the day of baking was characterized by the bread with the addition of barley preparation. The fastest rate of staling was observed in the bread with additional barley flour that was affected by the highest amount of β-glucan. A significant decrease of the β-glucan level was also found during the technological bake-off process, which can be explained by the activity of enzymes.