Background: Migration, production of reactive oxygen species (ROS), release of myeloperoxidase (MPO), and NETosis are functional immunological reactions of elementary importance for polymorphonuclear neutrophils (PMN). Unregulated inflammatory response of PMN within tissues plays a key role in the pathophysiology of several diseases. However, little is known about the behavior of PMN after migration through blood vessel walls. Therefore, we investigated the influence of the extracellular matrix (ECM) on PMN function. Materials and Methods: We established an in vitro chemotaxis model of type I and III collagen, fibrin, and herbal agarose tissues using mu-slide chemotaxis devices and N-formylmethionine-leucyl-phenylalanine (fMLP). PMN within the matrices were assessed with a fluorescent time-lapse microscope for live-cell imaging. Results: PMN function was obviously influenced by the ECM. Type III collagen had an inhibitory effect on PMN migration regarding track length, direction, and targeting. Type III collagen also had an accelerating effect on neutrophil ROS production. Agarose had an inhibitory effect on MPO release and fibrin a retarding effect on NETosis. Conclusion: Because of the high abundance of type III collagen in lung and skin matrices, the interaction of PMN with the respective matrix could be an important mechanism in the pathophysiology of acute respiratory distress syndrome and pyoderma gangrenosum.