AbstractInnate immunity is the first line of defense against infectious intruders and also plays a major role in the development of sterile inflammation. Direct microscopic imaging of the involved immune cells, especially neutrophil granulocytes, monocytes, and macrophages, has been performed since more than 150 years, and we still obtain novel insights on a frequent basis. Initially, intravital microscopy was limited to small‐sized animal species, which were often invertebrates. In this review, we will discuss recent results on the biology of neutrophils and macrophages that have been obtained using confocal and two‐photon microscopy of individual cells or subcellular structures as well as light‐sheet microscopy of entire organs. This includes the role of these cells in infection defense and sterile inflammation in mammalian disease models relevant for human patients. We discuss their protective but also disease‐enhancing activities during tumor growth and ischemia‐reperfusion damage of the heart and brain. Finally, we provide two visions, one experimental and one applied, how our knowledge on the function of innate immune cells might be further enhanced and also be used in novel ways for disease diagnostics in the future.