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These immune cells are recruited to infectious sites to reach and phagocytose pathogens. Specifically, in this article, bacteria from the genus <jats:italic>Mycobacterium<\/jats:italic>, <jats:italic>Salmonella<\/jats:italic> and <jats:italic>Escherichia<\/jats:italic>, were selected to study the directional macrophage movement towards different bacterial fractions. We recreated a three-dimensional environment in a microfluidic device, using a collagen-based hydrogel that simulates the mechanical microarchitecture associated to the Extra Cellular Matrix (ECM). First, we showed that macrophage migration is affected by the collagen concentration of their environment, migrating greater distances at higher velocities with decreasing collagen concentrations. To recreate the infectious microenvironment, macrophages were exposed to lateral gradients of bacterial fractions obtained from the intracellular pathogens <jats:italic>M. tuberculosis<\/jats:italic> and <jats:italic>S. typhimurium<\/jats:italic>. Our results showed that macrophages migrated directionally, and in a concentration-dependent manner, towards the sites where bacterial fractions are located, suggesting the presence of attractants molecules in all the samples. We confirmed that purified <jats:italic>M. tuberculosis<\/jats:italic> antigens, as ESAT-6 and CFP-10, stimulated macrophage recruitment in our device. Finally, we also observed that macrophages migrate towards fractions from non-pathogenic bacteria, such as <jats:italic>M. smegmatis<\/jats:italic> and <jats:italic>Escherichia coli<\/jats:italic>. 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