Circadian rhythms play an important role in maintaining the proper regulation of cognitive function and physiological processes. The importance of this system is highlighted when desynchronization of circadian rhythms occurs; however, the process by which these changes to brain and behavior remains unknown. Microglia cells are a possible mechanism for these changes because they are regulated by circadian rhythms and directly and indirectly influence synapses. In order to understand the role of time-of- day and perturbations of circadian rhythms on microglia morphology and function, a dual approach, using transcriptomics and multiplexed immunohistochemistry with imaging, was implemented. We observed an effect of time-of-day (ZT4 vs ZT16) on a range of functional pathways, including genes belonging to the “Activated Microglia” gene set, in the PFC of male and female mice. We demonstrated an increase in phagocytic capacity at ZT0 compared to ZT12. Under a T20 model of circadian desynchronization, a general upregulation in differentially expressed genes is visualized. When analyzing the combination of transcriptomics and morphological characteristics of microglia under T20 conditions, an overall more “activated” phenotype with higher phagocytic capacity was observed.