Objective Red soil is a typical soil in Southern China, and organic substitution is an important way to improve the physicochemical properties of red soil. This study aimed to explore the effects of different organic substitution materials and aggregation on the microfauna communities in a red soil.Methods Soil samples from five long-term fertilization treatments were collected, including application of inorganic nitrogen, phosphorus, and potassium (NPK) fertilizer alone (I), NPK+peanut straw (IPS), NPK+rice straw (IRS), NPK+radish (IR) and NPK+pig manure (IPM). Soil aggregates were fractionated, and high-throughput sequencing was used to determine the microfauna communities within the aggregates.Results Compared to I, IPM and IRS significantly increased soil organic matter content, while IPS and IR had relatively smaller effects; IPM also significantly increased soil pH, total nitrogen, and available phosphorus content. Organic substitution had no significant effect on the diversity of soil microfauna but significantly influenced their community structure. Nematodes were the most abundant soil microfauna in upland red soil, with a relative abundance of 93.0% in I; IPM and IRS significantly reduced their relative abundance to 62.2% and 70.0%, respectively, while IPS and IR had smaller effects. Aggregate size significantly affected the Shannon and Chao1 indices of soil microfauna, with the diversity indices of the 53—250 μm and < 53 μm aggregates significantly higher than those of the 250—2 000 μm and > 2 000 μm aggregates, indicating that smaller particle sizes were more conducive to increasing soil microfauna diversity. Aggregate size also significantly affected soil microfauna community structure, but its impact was weaker than that of organic substitution.Conclusion Both organic substitution and aggregate size could significantly affect soil microfauna communities, with pig manure and rice straw having greater effects than peanut straw and radish. The aggregation process reduced microfauna diversity in red soil.