Soil carbon (C) sequestration has been identified as a means to mitigate global climate change. Soil fungi, especially arbuscular mycorrhizal fungi (AMF), can influence soil biophysical characteristics important in sequestering C in soils. Fungicides and phosphorus have been used in studies involved in assessing soil C allocation and dynamics. This study examined the influence of suppressing soil AMF root colonization which influences soil structure, C allocation and nutrient dynamics, and hence ecosystem potential to accumulate C. The study area was located at the Konza Prairie Biological Station. The experimental design was a split plot with main plots arranged in a randomized complete block. Main plots were native prairie grass (big bluestem; PG), and no till (NT) grain sorghum and tilled (CT) grain sorghum. Treatments to suppress AMF included phosphorus (P), fungicide (F), and fungicide and phosphorus (F+P). A control for each main plot was included in the design (n = 4). The fungicide was Topsin® 70 WP; P was applied at a rate of 90 kg P ha-1. The P treatment was initiated in 2004 and F in 2005 and both applied annually until 2007. Samples were collected in 2008. Percent AMF root colonization was estimated using the magnified gridline intersect method. Chloroform fumigation-incubation method was used to estimate microbial biomass C. Soils were separated into aggregate size classes by wet sieving whereby macroaggregates was defined as >2000 µm and 250-2000 µm while microaggregates were 53-250 µm and 20-53 µm. Total soil organic C (TOC) and aggregate C were estimated using dry combustion. Percent AMF root colonization and MBC were significantly greater in PG than NT and CT. Relative to the controls, the reduction of % AMF root colonization in PG was F+P > F > P, NT was F+P > F > P , and CT was F > P > F+P . While the P amendment alone did not reduce TOC, F+P and F treatments reduced TOC in PG and NT relative to their respective controls. Formation of water-stable macroaggregates was PG > NT > CT and the reverse was true for microaggregates. There was more TOC associated with the macroaggregates than the microaggregates. These results support our prediction that AM fungi are critical to the formation of maccroaggregates and soil C sequestration.