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Soil micro-food webs play an essential role in maintaining or improving the stability of agricultural soils, and they can be influenced by tillage. However, little is known with respect to soil microbial and faunal communities and their relationships shaped by long-term tillage practices. The goal of this study was to investigate the impact of 38 years of no-tillage (NT), subsoil tillage (ST), moldboard plow tillage (MP), and rotary and ridge tillage (CT) practices on soil microbial and faunal communities, and their relationships with soil properties using high-throughput sequencing technology and structural equation modeling (SEM) at 2 soil depths (0–20 cm and 20–40 cm). The results indicate that, after the 38-year (1983–2020) period, the bacterial, fungal, protozoan, and metazoan gene copy numbers under the NT treatment at 0–20 cm were 1.31–6.13 times higher than those under the other treatments. Conversely, the microbial and protozoan alpha diversities were reduced under the NT treatment compared with the CT treatment. However, MP significantly increased microbial and faunal gene copy numbers at 20–40 cm. Moreover, the bacterial community composition remarkably varied relative to the community composition of the fungi and fauna in response to the tillage practices and soil depths. Additionally, the highest and lowest average connectivities of the soil micro-food web networks were observed under the ST and MP treatments, respectively. The SEM demonstrated that tillage practices and soil depths explained 73–98% of the microbial and faunal abundances, diversities, and compositions. Additionally, tillage and depth demonstrated direct quantitative effects and indirect quantitative effects by altering the soil mean weight diameter of aggregates, soil organic carbon, and total nitrogen. Overall, subsoil tillage is recommended as the optimal practice for application in northeast China, and it could improve soil properties and aid in forming a more complex soil micro-food web structure.