Abstract Aggregation is important for soil functioning, providing physical protection of organic matter and microbial inhabitants. Tillage disrupts aggregates, increases wind and water erosion of soils and exposes formerly protected organic matter to decomposition and losses. Microbial biomass and community dynamics in dry-sieved aggregate-size classes from long-term no-till (NT) and conventionally tilled (CT) soils were examined using phospholipid fatty acid analysis (PLFA). Bacterial, fungal, and total biomass were up to 32% greater in NT compared to CT aggregates. Aggregate size also affected microbial biomass, which was highest in the 1–2 mm size class. Arbuscular mycorrhizal fungi (AMF) were particularly affected by tillage disturbance with increases of 40–60% among aggregate-size classes in NT vs. CT, but glomalin related soil protein concentration was not different between tillage treatments or among aggregate-size classes. Bacterial stress biomarkers were higher in CT than NT aggregates but were not significantly correlated with total C, total N or C:N ratio, indicating that the physiological status of bacteria within aggregates was not simply governed by the quantity of available resources. Ordination analysis of PLFA profiles demonstrated a shift in microbial community structure between NT and CT aggregates, correlated with AMF abundance in NT aggregates and increased bacterial stress biomarkers in CT aggregates. Our results demonstrated greater microbial biomass and altered microbial community structure in NT vs. CT aggregates. This work demonstrates that tillage management influences microbial community structure within aggregates and may provide a potential explanation for differences in process rates observed in NT vs. CT soils. Further research into the processes that govern community structure in aggregates from NT and tilled soils is needed to better understand how the interaction of microorganisms with their physical environment affects nutrient turnover and availability.