Elemental sulfur is widely used to acidify alkaline calcareous soils and enhance nutrient availability, particularly in semi-arid regions where soil fertility is a limiting factor for crop production. This study investigates the effects of elemental sulfur (S) applications on soil microbial activity in alkaline calcareous soils, prevalent in dry and semi-arid subtropical regions. Powdered sulfur was sprinkled on the plot surface and mixed into the soil with a rotovator. Utilizing a randomized block design, three treatments were established: a control group, and two sulfur application rates (100 kg and 200 kg per decare). Each treatment was replicated three times in a randomized block design. Soil samples were collected five months post-application to assess microbial biomass (Cmic), basal soil respiration (CO2-C), and enzyme activities (acid, alkaline phosphatases, and dehydrogenase). Sulfur application at 100 kg/da caused moderate reductions in microbial biomass and enzyme activities, while 200 kg/da led to substantial declines, with microbial biomass (Cmic) and respiration rates (CO2-C) decreasing by 54% and 38%, respectively. Additionally, enzyme activities decreased by 19% for alkaline phosphatase, and 23% for dehydrogenase at the highest sulfur application rate. These findings suggest that excessive sulfur applications can negatively impact soil microbial communities and enzyme activities.