This study investigated relationships between ecoenzymatic stoichiometry and microbial community structure under long-term (38-years) combined organic and inorganic fertilization. Experiment was initiated in 1982 and seven fertilization treatments were studied that included: CK (no fertilizer), NPK (inorganic N, P and potassium fertilizers), NPM (inorganic N, P fertilizers and manure), NKM (inorganic N, K fertilizers and manure), PKM (inorganic P, K fertilizers and manure), NPKM (inorganic N, P, K fertilizers and manure) and M (manure) treatments. We analyzed soil and microbial biomass of C, N and P, enzyme activities related to C, N and P, and their effects on the microbial community structure in the top layer (0–20 cm) of the paddy soil. The results showed that combined application of manure and inorganic fertilizers significantly increased soil C, N, P contents, extracellular enzyme activities and total phospholipid fatty acid (PLFA) concentration, compared to the NPK and CK treatments. Fertilization treatments significantly changed microbial biomass stoichiometry. Among all fertilization treatments, highest microbial biomass C:N, C:P ratio and N:P ratios were 10.2, 36.0 and 3.59, respectively, under the PKM treatment. Soil C, N and P-cycling enzyme activities were highest under the NPKM treatments. Soil C:N showed positive and soil C:P ratio showed negative relationships with microbial biomass C:N and C:P ratios, respectively. Soil total PLFA concentration was highest under NPKM treatment. The order of individual PLFAs concentrations in all fertilization treatments was: Gram negative bacteria > Gram positive bacteria > Actinomycetes > AM fungi > Fungi> Eukaryotes > anaerobes. Soil organic C, total N, soil N:P and microbial N:P ratios were positively correlated with total PLFA concentration. While, soil C:N and microbial biomass C:N ratios were negatively correlation with total PLFA and individual microbial community structure. Our results suggested that ecoenzymatic stoichiometry and microbial community structure are very sensitive to combined long-term organic and inorganic fertilization that can affect the nutrient acquisition and cycling in agroecosystem.