Microbial biomass and its activity influence soil quality to sustain crop productivity. A field experiment was conducted to investigate changes in soil microbial community structure, enzyme activities, and associated biological properties in response to biochar application in a tropical ecosystem. The treatments included control/no biochar (CT), 15-ton biochar ha−1 (BC-15), 30-ton biochar ha−1 (BC-30), and 30-ton corn cob biochar ha−1 + phosphate fertilizer (BC-30 + P). Biochar significantly increased microbial biomass C (by 4.5 to 8.2 folds) and N (by 1.4 to 2.7 folds), and mineralized C (by 1.2 to 1.7 folds). Incorporation of biochar at 30-ton ha−1 equally resulted in an efficient soil microbial activity by significantly decreasing specific maintenance respiration (qCO2) rates by 66 to 73%. Dehydrogenase and urease enzyme activities were increased by biochar amendments. Biochar treatments increased the abundance of arbuscular mycorrhiza (AMF) and soil fungi. However, only the BC-30 and BC-30 + P treated soils recorded increases in Gram-positive (G+) bacteria by 1.7 to 1.9 folds, and Gram-negative (G−) bacteria by 1.5 to 1.6 folds, respectively. For all functional groups of microbial communities, which included AMF, G+/G− bacteria, fungi, and actinobacteria, the highest concentration of total phospholipid fatty acid was observed when the soils were treated with 30-ton biochar ha−1. The most important soil factors that greatly contributed to the changes in microbial community composition were soil basal respiration, urease and dehydrogenase enzymes, microbial biomass carbon and nitrogen, metabolic quotient, and pH. The high fungal: bacteria in the soils treated with 30-ton biochar ha−1 implies that corn cob biochar amendment could increase C sequestration potential and enhance ecosystem stability in weathered tropical soils.