Objectives Organic farming has been widely promoted as an environmentally friendly production system, however, its long-term impact on farmland ecosystems remains unclear. Based on a 14-year long-term field experiment, this study investigated the soil microbial community structure and functions under organic farming, aiming to provide scientific evidence for the sustainable and high-quality development of organic agriculture.

Methods The long term field experiment was conducted in Changzhou, China. Soil samples were collected in topsoil (0−20 cm) and subsoil (20−40 cm) under organic and conventional farming systems. Soil physicochemical properties and enzyme activities were measured, while microbial community diversity was assessed using high-throughput sequencing. Redundancy analysis (RDA), correlation heatmaps, and microbial co-occurrence network analysis were applied to elucidate the effects of organic farming on soil microbial community structure, functional traits, and their interactions with environmental factors.

Results Compared with conventional farming, long-term organic farming significantly altered soil physicochemical properties and enzyme activities. In the 0–20 cm soil layer, soil organic matter (SOM), electrical conductivity (EC), available nitrogen (AN), and available phosphorus (AP) decreased significantly (P < 0.05), with reductions ranging from 14.8% to 51.2%. In the 20–40 cm soil layer, AN and microbial biomass carbon (MBC) decreased significantly by 46.2% and 62.9%, respectively, whereas dissolved organic carbon (DOC) increased significantly by 25.2%. Linear mixed-effects model (LMM) analysis revealed significant interactions between farming system and soil depth for AP, AN, and MBC (P < 0.05). Organic farming reshaped the structure and composition of soil microbial communities, with fungal α-diversity showing a higher trend compared to conventional farming. In bacterial communities, the relative abundances of Acidobacteriota and Nitrospirota in the 0−20 cm soil layer increased by 14.8% and 34.7%, respectively, while Bacillota and Bacteroidota in the 20−40 cm layer increased by 40.5% and 110.7%. In fungal communities, the relative abundances of Basidiomycota and Rozellomycota in the 0−20 cm layer increased by 24.6% and 174.7%, respectively, while Ascomycota and Rozellomycota in the 20−40 cm layer increased by 13.8% and 277.2%. Microbial co-occurrence network analysis revealed that bacterial networks exhibited greater overall complexity and connectivity than fungal networks, whereas fungal networks displayed a more compact and tightly interconnected interaction structure. Bacterial networks under conventional farming were larger and more redundant, whereas organic farming enhanced fungal network connectivity and local clustering. Specifically, average connectivity, average path length, and network density of fungal networks increased by 59.5%, −22.6%, and 66.6%, respectively. Correlation analyses indicated that soil pH, SOM, and AP were the key environmental drivers of microbial community structure and composition. Bacterial communities exhibited stronger responses to environmental variations than fungal communities.

Conclusions Long-term organic farming altered soil physicochemical properties and enzyme activities, reshaping microbial community structure and functional traits. These changes resulted in enhanced fungal diversity, reduced bacterial network complexity, and increased fungal network connectivity. Moreover, functional predictions indicated a rise in potential pathogenic groups under organic farming, highlighting the need to consider disease risks in long-term organic systems.