Replenishing the soil with organic carbon regulates climate change and soil ecosystem functions. Limited information is available regarding the potential effect of different types of biochar on organic carbon mineralization and biochemical functions in alkaline subtropical soil. The study aimed to assess the impact of different biochars and their application rates on soil microbial and biochemical properties. An incubation experiment was conducted to evaluate the effects of biochars i.e., sugarcane filter cake biochar (SF-BC), farmyard manure biochar (FM-BC), and rice husk biochar (RH-BC), and their application rates (0, 0.25%, 0.5%, and 1%) on soil chemical and biochemical properties, including C mineralization. The incubation was carried out for 72 days at 22 C-o. Soil biochemical properties such as microbial biomass C, alkaline phosphatase activity, dehydrogenase activity, ergosterol, and amino sugars were significantly influenced by biochar types and increased with increasing application rates, maximum values were observed at 1% biochar application rate. FM-BC amended soil produced 16% and 9% more cumulative CO2 respectively compared to SF-BC and RH-BC amended soil, which linked to the labile C contents. Irrespective of biochar types, soil pH decreased to 7.81 and 7.78 with application rates of 0.50% and 1.00% respectively compared to the control (7.83). FM-BC amended soil showed the highest electrical conductivity i.e., 0.31 dS m(-1,) and higher contents of dissolved organic C, microbial biomass C, alkaline phosphatase activity, and ergosterol. The study suggests that FM-BC can enhance soil biochemical functions, while SF-BC and RH-BC can efficiently sequester carbon in organic carbon-deficient alkaline soil.