The stream bed is considered as an important key habitat, yet there is limited knowledge on the effects of burrowing organisms and the resulting bioturbation on this habitat. In this study, a standardized laboratory experiment was used to compare the impact of burrowing lamprey larvae (Eudontomyzon sp.) on physical and chemical habitat properties as well as microbial community composition in freshwater substrates to a control without animals. We hypothesized that the burrowing activity of the lamprey larvae increases the influx of open water into interstitial areas with consequences for oxygen availability and thus for microbial community composition. The study revealed a strong increase in oxygen availability (oxygen concentration and redox potential) and nitrate concentrations in interstitial water in the lamprey treatment, indicative of an increased exchange with open water. Over time, a shift in microbial community composition was observed in the lamprey treatment, which was dominated by aerobe bacteria. Because the functional relevance of lamprey larvae for freshwater ecosystems is evidentially high, they can clearly be considered as ecosystem engineers. Their important ecosystem functions should be recognized in conservation and management plans.