Glomalin has been suggested as an enhancer for soil stability by promoting the aggregation. In this study, we examined the concentrations of glomalin and microbial characteristics in 25 controlled horticultural soils sampled from Gyeongnam Province. Total glomalin had a significant positive correlation with soil organic matter (p < 0.01), soil microbial biomass carbon (p < 0.05), and dehydrogenase activity (p < 0.05) in controlled horticultural soils. In addition, the total glomalin had a significant positive correlation with concentrations of total fatty acid methyl esters, Gram-negative and Gram-positive bacteria, fungi, and arbuscular mycorrhizal fungi in controlled horticultural soils (p < 0.001). In conclusion, the concentration of total glomalin could be an indicator of microbial biomass richness for sustainable agriculture in controlled horticultural soils.