Down syndrome (DS) is characterized by deficits in neuronal activity and connectivity, with unclear contributions from different cell types. Microglia, the brain's immune cells, shape neuronal circuits by influencing the number of synaptic connections, both excitatory and inhibitory. This study investigates the microglial involvement in DS-related connectivity deficits. Using in vivo mesoscale wide-field calcium imaging, we analyzed the cortical connectivity of Ts65Dn and euploid mice injected retroorbitally with a CAMKII-GCaMP7f viral vector, in response to a drug effective in blocking neuron-to-microglia communication, a P2Y12R inhibitor. Baseline assessment revealed profound connectivity deficits in Ts65Dn mice. Subsequent drug administration induced broad hypoconnectivity in controls but not in trisomic mice, highlighting not only the importance of microglia in determining connectivity properties in the healthy brain but also unveiling dysregulated neuroimmune signaling in DS. Immunofluorescence analysis of cortical areas showed how microglia in DS brains preferentially eliminate inhibitory puncta over excitatory synapses compared to controls. Finally, by deriving primary neurons and microglia from either trisomic or euploid mice, we demonstrate how DS microglia exert distinct effects on inhibitory and excitatory connectivity and activity compared to euploid microglia. Of note, engulfment of inhibitory puncta by microglia is strongly influenced by microglial genotype - DS microglia preferentially prune more inhibitory puncta compared to euploid - while the loss of excitatory puncta appears to be solely driven by disease-carrying neurons. Moreover, DS microglia selectively impact neuronal calcium activity in vitro. Our findings suggest that microglia contribute to DS by differentially influencing excitatory and inhibitory circuits.