We examined the effect of reduced tillage on the accumulation of fungal‐ versus bacterial‐derived organic matter within the soil matrix by quantifying the amino sugars glucosamine (Glc), galactosamine (Gal), and muramic acid (MurA) in aggregate‐size fractions isolated from no‐tillage (NT) and conventional‐tillage (CT) soil. Intact soil cores (0‐ to 5‐ and 5‐ to 20‐cm depth) were collected from the long‐term tillage experiment at Horseshoe Bend in Athens, GA. Four water‐stable aggregate‐size fractions were isolated: large macroaggregates (>2000 μm), small macroaggregates (250–2000 μm), microaggregates (53–250 μm), and the silt + clay fraction (<53 μm). Small macroaggregates were further separated into coarse particulate organic matter (POM) (>250 μm), microaggregates contained within macroaggregates, and the silt + clay fraction. Amino sugars were extracted from all fractions, purified, and analyzed by gas chromatography. Fungal‐derived amino sugar C (FAS‐C) comprised 63%, while bacterial‐derived amino sugar C (BAS‐C) accounted for 37% of the total amino sugar C pool under both tillage treatments. No‐tillage soil contained 21% more amino sugar C than the CT soil across the entire plow layer. Both, the percentage of total organic C as FAS‐C and BAS‐C were significantly higher in the silt + clay fraction of NT versus CT soil. The percentage of total organic C as FAS‐C was significantly higher in small macroaggregates of NT versus CT soil due to a preferential accumulation of FAS‐C in the microaggregates contained within these macroaggregates. These results indicate that microbial‐derived C is stabilized in NT soils, due primarily to a greater fungal‐mediated improvement of soil structural stability and concurrent deposition of fungal‐derived C in microaggregates contained within macroaggregates.