Microbial communities residing in forest soils play crucial roles in decomposing organic matter and recycling nutrients, making these ecosystems one of the most diverse habitats on Earth. However, the composition and function of these complex and diverse microbiomes across different soil layers remain largely unknown. In this study, we collected soil samples from various layers and analysed the bacterial and fungal community compositions in experimental forest ecosystems using sequencing techniques. Our findings revealed that the soil layer was the primary factor influencing microbial communities, whereas sampling season had only a marginal effect. The most prevalent bacterial phyla and fungal classes were Acidobacteria, Actinobacteria, Armatimonadetes, Bacteroidetes, Firmicutes, Planctomycetes, Proteobacteria, Verrucomicrobia, and Agaricomycetes. Owing to the heterogeneity of the soil layer environment, we observed distinct patterns in the bacterial and fungal microbiomes across different layers. Moreover, the soil layer affected the network complexity, with fungi exhibiting higher complexity in the upper layer, whereas bacteria showed the opposite trend. Additionally, the dominant bacterial and fungal taxa across all soil layers belonged predominantly to Acidobacteria and Agaricomycetes, respectively. These findings underscore the significance of soil layers in shaping soil microbial communities and highlight the composition and co-occurrence patterns of the microbial communities within these layers.