Increasing the abundance of fungi relative to bacteria should favor C accrual, because fungi use C more efficiently, and are composed of more recalcitrant C compounds. We examined changes in soil microbial community structure following cessation of tillage-based agriculture and through subsequent succession in a C-accruing tallgrass prairie restoration chronosequence. We predicted that the relative abundance of fungi would increase following conversion from tillage-based agriculture. Soil microbial community structure was assessed as relative abundances of phospholipid fatty acids (PLFAs). Cessation of tillage-based agriculture did initially lead to an increase in the abundance of fungi, particularly arbuscular mycorrhizal fungi (AMF), relative to bacteria. We suggest this is primarily due to reduced disturbance when tilling ceases. Vegetation characteristics also appear to be important, with high cyclopropyl/precursor PLFA ratios indicating bacterial communities under stress in agricultural soils, probably due to low C, and possibly to low C relative to N inputs. A secondary gradient in soil microbial community structure was related to successional time, and tied to soil characteristics, particularly bulk density (Db), pH, and soil organic C and N. However, while the fungi/bacteria (F/B) ratio was high in early succession plots, it declined later in succession. In addition, although the F/B ratio increased with SOC in the agricultural soils, it decreased with SOC in prairie soils. We conclude that increased community metabolic efficiency due to higher relative abundances of fungi is not the primary mechanism leading to enhanced C storage in these soils.