The sensitivity of creeping bentgrass (Agrostis palustris Huds.) to the extreme heat found in the southeastern United States has led to the development of new greens-management methods. The purpose of this study was to examine the effects of subsurface aeration and growth regulator applications on soil microbial communities and mycorrhizal colonization rates in a creeping bentgrass putting green. Two cultivars (Crenshaw and Penncross), a growth regulator (trinexapacethyl), and subsurface aeration were evaluated in cool and warm seasons. Total bacterial counts were higher in whole (unsieved) soils than in sieved soils, indicating a richer rhizosphere soil environment. Mycorrhizal infection rates were higher in trinexapac-ethyl (TE) treated plants. High levels of hyphal colonization and relatively low arbuscule and vesicle occurrence were observed. Principal components analysis of whole-soil fatty acid methyl ester (FAME) profiles indicated that warm-season microbial populations in whole and sieved soils had similar constituents, but the populations differed in the cool season. FAME profiles did not indicate that subsurface aeration and TE application affected soil microbial community structure. This is the first reported study investigating the influences of subsurface aeration and TE application on soil microorganisms in a turfgrass putting green soil.