Adding manure to croplands restores carbon and nutrients in depleted soils, while addressing a waste disposal need. This practice depends on the abundance and activity of microbial communities to break down manure inputs, which provide carbon for microbial growth and release nutrients for plant and microbial uptake. In a 2-year field study, we measured changes to soil physicochemical properties and microbial community composition in a conventional rotation of summer sileage corn and winter wheat. Three ratios of manure to mineral N fertilizer were used: all manure (100% of plant N from manure), mixed (50% plant N from by manure and 50% from mineral fertilizer), and all mineral (100% plant N from mineral fertilizer). Yields of corn and wheat were similar across treatments. Soil microbial community dissimilarity increased with higher ratios of application, suggesting a dose-dependent relationship between manure application and microbial community composition. Both microbial biomass and dissolved organic carbon showed the greatest response immediately following manure application, then decreased until the next application. The Shannon index increased shortly after manure application, with the largest increase observed in soils receiving manure input. Many of the taxa that increased in relative abundance following manure application were specifically taxa known to be present in manure and not common or present in soil before manure application. Microbial community changes following manure application were transitory, indicating that without continual inputs of resources, these alterations are not maintained. This was in contrast to microbial biomass which slowly increased in size over the 2-year study period. Better understanding of how manure interacts with microbial communities will help in developing better nutrient management practices and help reduce our reliance on mineral fertilizers.