Past land use can impart soil legacies that have important implications for ecosystem function. Although these legacies have been linked with microbially mediated processes, little is known about the long-term influence of land use on soil microbial communities themselves. We examined whether historical land use affected soil microbial community composition (lipid profiles) and whether community composition was related to potential net nitrogen (N) mineralization rates in southern Appalachian (USA) forest stands abandoned from agriculture or logging and reforested >50 yr ago. Microbial community composition was determined by a hybrid procedure of phospholipid fatty acid (PLFA) and fatty acid methyl ester (FAME) analysis. We found that community composition varied significantly with past land use. Communities in formerly farmed stands had a higher relative abundance of markers for gram-negative bacteria and a lower abundance of markers for fungi compared with previously logged and reference (i.e., no disturbance history) stands. Potential net N mineralization rates were negatively correlated with fungal and gram-negative bacterial markers in both farmed and reference stands, and fungal abundance and soil bulk density effectively predicted mineralization rates in all stands. Our results indicate that the alteration of microbial communities by historical land use may influence the ecosystem processes they mediate. This is in contrast to typical expectations about microbial community resilience to change. Here, the decrease in fungal abundance observed from disturbance appeared to result in decreased nitrogen mineralization over the long term.