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Continuous cultivation has been known to decrease soil organic matter content. Application of organic matter to cultivated soil is an important practice from the point of view of maintaining an adequate amount of soil organic matter. Soil organic matter content significantly affects soil microbial activity, which is an important index of soil quality. In this study, a field experiment was conducted to examine the long-term effects of different kinds of organic matter in combination with inorganic nitrogen (N) fertilizer on chemical and biological properties of soils. There were seven treatments, namely (1) The results showed that compared with CK and Chem-N treatments, applications of compost and peat increased soil organic carbon (SOC) content and altered microbial activities and microbial community structure. However, application of green manure for 12 years had no effect on SOC content. Both microbial activities and PLFA profiles were clearly dependent on the characteristics of the applied organic amendments. In summary, a peat application led to the highest increase in SOC content compared to compost and green manure; however, compost-treated soil had a higher microbial population and higher microbial and enzyme activities, while the effects of both green manure and chemical N fertilizer on soil properties were similar.