Cover cropping is a widely promoted strategy to enhance soil health in agricultural systems. Despite a substantial body of literature demonstrating links between cover crops and soil biology, an important component of soil health, research evaluating how specific cover crop species influence soil microbial communities remains limited. This study examined the effects of eight fall-sown cover crop species grown singly and in multispecies mixtures on microbial community structure and soil biological activity using phospholipid fatty acid (PLFA) profiles and daily respiration rates, respectively. Fourteen cover crop treatments and a no cover crop control were established in August of 2011 and 2012 on adjacent fields in central Pennsylvania following spring oats (Avena sativa L.). Soil communities were sampled from bulk soil collected to a depth of 20 cm (7.9 in) in fall and spring, approximately two and nine months after cover crop planting and prior to cover crop termination. In both fall and spring, cover crops led to an increase in total PLFA concentration relative to the arable weed community present in control plots (increases of 5.37 nmol g−1 and 10.20 nmol g−1, respectively). While there was a positive correlation between aboveground plant biomass (whether from arable weeds or cover crops) and total PLFA concentration, we also found that individual cover crop species favored particular microbial functional groups. Arbuscular mycorrhizal (AM) fungi were more abundant beneath oat and cereal rye (Secale cereale L.) cover crops. Non-AM fungi were positively associated with hairy vetch (Vicia villosa L.). These cover crop-microbial group associations were present not only in monocultures, but also multispecies cover crop mixtures. Arable weed communities were associated with higher proportions of actinomycetes and Gram-positive bacteria. Soil biological activity varied by treatment and was positively correlated with both the size and composition (fungal:bacterial ratio) of the microbial community. This research establishes a clear link between cover crops, microbial communities, and soil health. We have shown that while cover crops generally promote microbial biomass and activity, there are species-specific cover crop effects on soil microbial community composition that ultimately influence soil biological activity. This discovery paves the way for intentional management of the soil microbiome to enhance soil health through cover crop selection.