The soil samples were taken from different treatments including CK, P, NP, NPK, MP（manure + P）, MNP（manure + NP）, and MNPK（manure + NPK） in a Mollisol long-term fertilizer experimental site. Basic soil properties, phospholipid fatty acid（PLFA）, acid（alkaline） phosphatase activities and soil microbial biomass C or N（SMB-C or N） were measured. The results showed that 26 years of the combined application of manure and chemical fertilizers had significantly increased the amounts of organic matter（OM）, total nitrogen（TN）, available phosphorus（AP）, alkali-hydrolysable nitrogen（AN）, and readily available potassium（RAK）. Furthermore, the SMB-C, SMB-C/organic matter（OM） ratio, acid（alkaline） phosphatase activities and PLFA content of different microbial communities in manure treatments were higher than those of CK or chemical fertilizer treatments. Long-term application of NPK treatment obviously inhibited microbial activity, while single P or NP treatment had little effect on this arable Mollisol microbial community. Monounsaturated fatty acids-to-normal saturated fatty acids（MONO/NSAT） ratios in manure treatments were significantly higher than those of chemical fertilizer treatments or CK, while terminally branched saturated fatty acids-to-monounsaturated fatty acids（TBSAT/MONO） ratios exhibited extremely significant negative correlation with MONO/NSAT ratios and changed in a totally opposite way. Principal components analysis of PLFA showed that manure and chemical fertilizers affected microbial communities’ structure in a very different way. The loading values for the individual PLFA showed that fungal 18:26,9 was significantly affected by chemical fertilizer treatments, while G+ i15:0, i17:0, i16:0 were liable to be affected by manure amendment.