Resident microglia and peripherally-derived monocytes differentially enhance and/or dampen immune-mediated pathology in neuroinflammatory disease. However, dissecting their precise roles in the inflamed brain and their contributions to pathology has historically been impeded by specific methods capable of accurately distinguishing these cell types. Here we used dimensional-reduction analysis on high-parameter cytometry data, to revise flow cytometric gating strategies. This enabled more precise identification of microglia to characterise their role in severe neuroinflammation, as modelled in West Nile Virus (WNV) encephalitis. We confirmed our devised gating strategy by depleting microglia with PLX5622, blocking the infiltration of peripherally-derived cells and tracking the phenotype of adoptively transferred monocytes in the CNS. Analysis of cells in suspension identified four microglial phenotypes in the homeostatic and infected brain which corresponded with phenotypes identified in tissue using imaging mass cytometry. Detailed kinetic analysis showed that microglia altered their morphology and proliferative status early in infection, whilst producing IL-12, downregulating core homeostatic proteins and dying later in infection. Microglia were also accompanied by microglia-like and Ly6Chi macrophages in the lethal progression of WNV-infection which upregulated ‘microglia-specific’ markers upon CNS entry. We further identified limitations in using enzymatic brain digestion and ‘microglia-specific’ markers in the identification of microglia in neuroinflammation.