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Soil enzymes and microorganisms are both important to maintaining good soil quality and are also sensitive to changes in agricultural management. The individual effects of tillage, straw incorporation and nitrogen (N) fertilization on soil enzymes and microflora have been widely acknowledged, but their interactive effect remains largely unknown. In a 5–year in–situ field study, effects of rotary (RTS) and plow tillage (PTS) practices with straw incorporation combined with three N fertilization levels (0 kg N ha–1, CK; 187 kg N ha–1, MN; 337 kg N ha–1, HN) on soil enzyme activities and microbial communities were assessed. Our results showed that the activities of β–glucosidase (βG), N–acetylglucosaminidase (NAG) and acid phosphatase (APH) were improved in RTS+MN. The bacterial and fungal abundances in RTS+MN and RTS+HN were 1.27–27.51 times higher than those in other treatment groups. However, the bacterial and fungal alpha diversities were enhanced in PTS+MN and PTS+CK compared with other treatments, respectively. Proteobacteria and Basidiomycota were the predominant phylum for the respective bacterial and fungal communities. Moreover, significant interactive effects were found in the fungal community composition, but only minor impacts were observed on the bacterial community composition. Soil water content and penetration resistance contributed more to the soil enzyme activity and microbial community than other soil properties investigated, whereas there was a significant positive correlation between βG and APH activities and microbial abundance. These findings can provide new insights into tillage with straw incorporation and N fertilization on maize cultivation in northeast China.