Microbial biomass, as part of the active pool of soil organic matter, is critical in decomposition of organic materials, nutrient cycling, and formation of soil structure. We evaluated chloroform fumigation-incubation with subtraction of a control (CFI/F-C) and without subtraction of a control (CFI/F) as methods to assess biological soil quality. Relationships between CFI/F and:potential C mineralization, particulate organic C, and soil organic C were stronger (r(2) = 0.86 +/- 0.07, n = 232) than those between CFI/F-C and the same soil C pools (r(2) = 0.25 +/- 0.09) in soils from Georgia. From published data, relationships of CFI/F with potential C mineralization and soil organic C were stronger than those of chloroform fumigation-extraction and substrate-induced respiration with these soil C pools. Effects of land management on biological soil quality using CFI/F were consistent with those determined using other soil C pools as response variables. However, land management effects on biological soil quality using CFI/F-C were either contrary to those using other soil C pools or not detectable because of greater inherent variability in CFI/F-C. Chloroform fumigation-incubation without subtraction of a control is a robust and reliable method to assess biological soil quality under a wide range of soil conditions.