Soil characteristics vary across land uses, and soil microbial communities are likely to respond to such variations. However, such responses are not well understood. In this study, we examined how specific changes in soil organic matter (SOM) and geochemical characteristics influenced the bacterial and fungal community composition and structure across land uses. Soil samples were collected from grazing, sugarcane, forest and banana land uses in the Johnstone River catchment (wet tropics), northeastern Australia. High-throughput sequencing of 16S rRNA gene and ITS amplicons were employed to characterise soil bacterial and fungal communities, respectively. Bacterial and fungal α-diversity did not differ across land uses, whereas strong shifts in their community composition and structure were evident. Patterns in microbial community composition were strongly (p < 0.01) associated with changes in soil total organic carbon and nitrogen (TOC and TN), TOC:TN ratio, hot-water extractable organic carbon (HWEOC) and soil geochemical characteristics (e.g., pH, Co, Mn, K, Mg and P contents). Distinct bacterial community composition was observed in forest soils, while fungal communities were similar in banana and forest soils compared with sugarcane and grazing. Overall, our results showed that SOM characteristics were the major driver on structuring soil microbial communities across land uses, while soil bacterial communities were more sensitive to variations in SOM and geochemical characteristics compared with fungi. Results from this study have implications for developing more targeted management practices across different land uses to improve soil health and ecosystem function in a wet tropical area.