Wetting agents are the primary tool used to control soil water repellency (SWR) and localized dry spot (LDS), especially on sand-based soils. However, the effect of repeated applications of wetting agents on soil microbial populations is unknown. This two-year field experiment investigated six wetting agents representing different chemistry effects on a creeping bentgrass (Agrostis stolonifera L.) putting green with existing SWR. Four out of the six wetting agents improved soil volumetric water content in the second growing season, while others showed no effect. This result was negatively correlated to the development of LDS, and positively correlated to occurrence of an air-borne turf disease. Soil microbial populations, determined by soil phospholipid fatty acid (PLFA) analysis, found that none of the treatments applied caused a shift in microbial populations between fungi and bacteria, or gram-positive and gram-negative bacteria. The stress indicators such as saturated to mono-unsaturated fatty acids were not affected by the wetting agents applied as well. However, the wetting agent that contains alkyl block polymers (ABP; Matador) with proven capability for removal of soil organic coatings showed inhibition of microbial populations at one evaluation timing. This result suggested a temporary restriction in soil carbon availability for soil microorganisms following repeated ABP application, which likely contributed to the elevated LDS development observed. Another wetting agent, a combined product of a nonionic surfactant plus acidifiers (NIS; pHAcid), which is designed to reduce inorganic carbonates while enhancing wetting, elevated all soil microbial populations tested at the end of the experiment, indicating a desirable improvement in soil health. However, repeated application of NIS did not reduce SWR at the conclusion of this experiment, which, in combination with a previous report, suggested a minimal disturbance of soil organic coatings of the hydrophobic sand. Overall, this experiment suggested that soil microbial populations can be affected by wetting agents which may further influence SWR, yet the actual effect on soil microorganisms varies depending on the chemistry of the wetting agents.