Microglial cells are critical for glioma growth and progression. However, only little is known about intratumoral microglial behavior and the dynamic interaction with the tumor. Currently the scarce understanding of microglial appearance in malignant gliomas merely originates from histological studies and in vitro investigations. In order to understand the pattern of microglia activity, motility and migration we designed an intravital study in an orthotopic murine glioma model using CX3CR1-eGFP(GFP/wt) mice. We analysed the dynamics of intratumoral microglia accumulation and activity, as well as microglia/tumor blood vessel interaction by epi-illumination and 2-photon laser scanning microscopy. We further investigated cellular and tissue function, including the enzyme activity of intratumoral and microglial NADPH oxidase measured by in vivo fluorescence lifetime imaging. We identified three morphological phenotypes of tumor-associated microglia cells with entirely different motility patterns. We found that NADPH oxidase activation is highly divergent in these microglia subtypes leading to different production levels of reactive oxygen species (ROS). We observed that microglia motility is highest within the perivascular niche, suggesting relevance of microglia/tumor blood vessel interactions. In line, reduction of tumor blood vessels by antivascular therapy confirmed the relevance of the tumor vessel compartment on microglia biology in brain tumors. In summary, we provide new insights into in vivo microglial behavior, regarding both morphology and function, in malignant gliomas.