The function and integrity of nervous system require interactive exchanges among neurons and glial cells. Exosomes and other extracellular vesicles (EVs) are emerging as a key mediator of intercellular communication, capable to transfer nucleic acids, proteins and lipids, influencing numerous functional and pathological aspects of both donor and recipient cells. The immune response mediated by microglia derived exosomes are most prominently involved in the spread of neuroinflammation, neurodegenerative disorders and brain cancer. Therefore, in present study we describe a reproducible and highly efficient method for yielding a purified primary microglia cells followed by exosomes isolation and their characterization. In vitro biological assay demonstrates that microglia derived exosomes tested on 3D spheroids glioma culture were capable to inhibit the tumor invasion in time course. These results evidence that brain microglia derived exosomes could be used as nanotherapeutic agents against glioma cells.