Global warming has caused a series of ecological issues in terrestrial ecosystems, which will be further aggravated by increasing global average temperatures. Changes in altitude gradients are an important aspect of studying the effects of climate warming on terrestrial ecosystems. Currently, studies on the effect of altitude gradient on microorganisms have not yielded conclusive results mainly because the influence of vegetation types has not been considered. Therefore, in this study, Pinus taiwanensis at 1300, 1450, and 1600 m in the mid-subtropical Daiyun Mountain were used to study changes in soil microbial biomass and community structure at different altitudes. The results showed that soil total carbon, total nitrogen, total phosphorus, microbial biomass nitrogen (MBN), microbial biomass phosphorus (MBP), arbuscular mycorrhizal fungi (AMF), gram-negative fungi (GN), fungi, total phospholipid fatty acid (T_PLFA), and bacteria:fungi (F:B) ratio all decreased significantly with altitude. However, the gram-positive bacteria:gram-negative bacteria (GP:GN) ratio increased significantly with elevation. Redundancy analysis (RDA) showed that the temperature (T) and dissolved organic nitrogen (DON) content were the most important environmental factors that determined the soil microbial community structure. Studies have shown that compared with 1600 m altitude, the temperature at 1300 m altitude was higher, which promoted the mineralization of soil organic matter, and increased the availability of nutrients and number of microbial biomass in the soil, thereby increasing microbial biomass such as fungi and bacteria. Therefore, future climate warming will likely affect the composition of microbial communities in the region by changing the soil carbon, nitrogen, and phosphorus nutrient contents. These results are of great importance to further understand the effect of climate change on soil nutrient cycling in mountain ecosystems.