Soil samples were collected from various treatments of a longterm fertilization experiment in Mollisol, and analyzed for variation of basic soil properties, phospholipids fatty acids (PLFAs), neutral lipid fatty acids (NLFAs), acidic (alkaline) phosphatase activities and soil microbial biomass C or N (SMB-C or N). Results show that 26 years of combined application of organic manure and chemical fertilizers significantly increased the contents of OM, TN, and readily available N, P and K, as well as the values of SMB-C or N, and SMB-C/OM ratio, and improved phosphatase activity and PLFA content of various microbial communities. The activity of soil microbe in Treatment N was obviously lower than in CK, whereas Treatment P was the lowest in effect on activity and community structure of soil microbes. Variation of the relative NLFA content in various soil bacteria was just contrary to that of living microbial biomarkers, suggesting that in Treatments MN, MP and MNP, rich in nutrient content, hypopodes of various bacterial communities or accumulation of storage substances was limited. PCA of PLFA, NLFA and NLFA/PLFA revealed that the community structure of soil microbe varied sharply between organic manure treatments and chemical fertilizer treatments Analysis of the loading values of some individual PLFAs or NLFAs showed that fungal C18∶2ω6,9 was significantly affected by manure application, while G+ iC15∶0, iC17∶0, iC16∶0 were apt to be affected by chemical fertilizer application. PCA also demonstrated that NLFA/PLFA ratio is more applicable than PLFA or NLFA in distinguishing treatments different in fertilization.