Arbuscular mycorrhizal fungi (AMF) and their product glomalin (GRSP) play a decisive role in the soil aggregation, affecting the carbon (C) dynamics in agroecosystems. Tillage affects the AMF activity and GRSP content, influencing the stability and the soil C forms as well. The aim of this study was to compare the effect of no tillage (NT) and conventional tillage (CT) on: i) arbuscular mycorrhizal hyphal length and GRSP content; ii) the nature of soil organic matter by means of physical fractionation (free particulate organic matter [fPOM]; occluded particulate organic matter [oPOM] and mineral-associated soil organic matter [Mineral]), as well as chemical fractionation (fulvic acid, humic acid and humin), and iii) the relationships between AMF parameters, soil carbon and water stable aggregates (WSA) in a Mollisol of Central Chile managed for 6 years under NT and CT using a wheat-corn rotation. Higher values in the AMF hyphal length, GRSP and WSA in NT compared with CT were observed. Significant relationships were found between GRSP and WSA (r = 0.66, p < 0.01) and total mycelium and GRSP (r = 0.58, p < 0.05). The total carbon increased 44% under NT compared with CT. The chemical fractionation showed percentage greater than 95% for humim in both treatments. Physical fractionation indicates that the higher part of the SOC (89.4 - 95.1%) was associated with the mineral fraction.