As an alternative for phospholipid fatty acid (PLFA) analysis, a simpler ester linked fatty acid (ELFA) analysis has been developed to characterize soil microbial communities. However, few studies have compared the two methods in forest soils where the contribution of nonmicrobial sources may be larger than that of microbial sources. Moreover, it remains unclear whether the two methods yield similar relationships of microbial biomass and composition with environmental variables. Here, we compared PLFA and ELFA methods with respect to microbial biomass and composition and their relationships with environmental variables in six oriental oak (Quercus variabilis) forest sites along a 1500-km latitudinal gradient in East China. We found that both methods had a low sample-to-sample variability and successfully separated overall community composition of sites. However, total, bacterial, and fungal biomass, the fungal-to-bacterial ratio, and the gram-positive to gram-negative bacteria ratio were not significantly or strongly correlated between the two methods. The relationships of these microbial properties with environmental variables (pH, precipitation, and clay) greatly differed between the two methods. Our study indicates that despite its simplicity, the ELFA method may not be as feasible as the PLFA method for investigating microbial biomass and composition and for identifying their dominant environmental drivers, at least in forest soils.