Soil microbial community plays important roles in altering ecological processes and biogeochemical cycles in ecosystems. However, little is known about how afforestation influences the diversity, composition, and ecological network of soil microbial community in karst regions. In this study, soil samples were collected from one farmland (FL) and three afforestation lands including one bamboo forest (BA), one landscape tree planting forest (LAT), and one orange orchard (ORO) in a karst region of Southwest China. The bacterial and fungal communities were characterized using the high‐throughput sequencing approach, and soil properties including soil organic carbon, pH, soil water content, and soil total and available nutrient contents were measured under different land use treatments. Results showed that conversion from FL to BA and LAT significantly reduced the Shannon diversity of bacterial community. At the phylum level (top 10), genus level (top 30), and operational taxonomic units (OTUs) level, afforestation from FL resulted in significant changes in nine phyla, 24 genera, and 31.32% OTUs of bacterial community, and in three phyla, 13 genera, and 11.62% OTUs of fungal community. The number of nodes, number of negative edges, connectivity, average degree, and relative modularity of the microbial network under afforestation lands decreased by 9.33%–18.66%, 47.98%–72.75%, 0.45%–5.93%, 14.73%–22.29%, and 6.46%–23.50%, respectively, compared with FL. The soil organic carbon, total potassium and total phosphorus were identified as the key soil properties affecting the microbial community. Compared with LAT and ORO, the changes in bacterial and fungal communities under BA were more obvious because of the higher contents in soil organic carbon (13.48%) and total potassium (27.18%). In conclusion, afforestation significantly changed compositions and ecological network complexity of soil microbial community in karst regions, and conversion from FL to BA had stronger influences on the changes of soil microbial community than other afforestation lands in Southwest China. These findings provide the context necessary to evaluate the responses of soil microbial community to land use changes in karst regions.