Soil microbial community composition, aggregation and organic matter (SOM) content can be markedly influenced by tillage and crop management practices. This study was undertaken to determine to what extent differences in populations of mycelial fungi in conventional (CT) and no-tillage (NT) soils contribute to soil aggregation and soil organic matter storage. Fungicide (Captan) and control treatments were established in long-term CT and NT plots on a well-drained Hiwassee sandy clay loam soil (clayey kaolinitc thermic Rhodic Kanhapludult). The effects of these treatments on total and FDA-active fungal hyphal lengths, total bacteria (0–15 cm) and in situ soil respiration rates were measured at approximately monthly intervals. Soil carbohydrates and water-stable aggregate (WSA) distributions were quantified on the final sample date. Surface soil (0–5 cm) of NT had more macroaggregates (> 250 μm diam) and 1.30 to 1.46 times higher densities of fungal mycelia as compared to CT soils. The higher populations of fungal mycelia corresponded to a nearly two-fold higher concentration of acid-hydrolysable carbohydrates, which were composed of proportionally more microbial- than plant-derived sugars. Treatment with the fungicide resulted in a 40% reduction in > 2000 μm WSA and lower concentrations of carbohydrates in NT surface soils, but had no significant effects in CT soils. The contributions of fungi to aggregate stability may represent an important biotically-regulated mechanism for the protection of soil organic matter and may help to explain the greater retention of soil organic carbon in NT than in CT soils.