Using a scheme of agricultural fields with progressively less intensive management (deintensification), different management practices in six agroecosystems located near Goldsboro, NC, USA were tested in a large-scale experiment, including two cash-grain cropping systems employing either tillage (CT) or no-tillage (NT), an organic farming system (OR), an integrated cropping system with animals (IN), a successional field (SU), and a plantation woodlot (WO). Microbial phospholipid fatty acid (PLFA) profiles and substrate utilization patterns (BIOLOG ECO plates) were measured to examine the effects of deintensification on the structure and diversity of soil microbial communities. Principle component analyses of PLFA and BIOLOG data showed that the microbial community structure diverged among the soils of the six systems.Lower microbial diversity was found in lowly managed ecosystem than that in intensive and moderately managed agroecosystems, and both fungal contribution to the total identified PLFAs and the ratio of microbial biomass C/N increased along with agricultural deintensification. Significantly higher ratios of C/N (P ＜ 0.05) were found in the WO and SU systems, and for fungal/bacterial PLFAs in the WO system (P ＜ 0.05). There were also significant decreases (P ＜ 0.05)along with agricultural deintensification for contributions of total bacterial and gram positive (G+) bacterial PLFAs.Agricultural deintensification could facilitate the development of microbial communities that favor soil fungi over bacteria.