The environmental impacts of herbicides on desirable plants and the soil biota are of public concern. The surfactants that are often used with herbicides are also under scrutiny as potentially harmful to soil biological systems. To address these concerns, we used two soils, a silt loam and a silty, clay loam from south central Missouri, to investigate the impacts of herbicides and surfactants on soil microbial communities using phospholipid fatty acid (PLFA) analysis. The surfactants used in this study were alkylphenol ethoxylate plus alcohol ethoxylate (Activator 90), polyethoxylate (Agri-Dex), and a blend of ammonium sulfate, drift reduction/deposition polymers and anti-foam agent (Thrust). The herbicides were glyphosate, atrazine and bentazon. Surfactants and herbicides were applied to soils at label rate, either alone or combined, to 4000g soil per pot. The two soils differed in history, texture, some chemical characteristics and several microbial community characteristics. A few of the chemicals altered some of the components of the microbial community after only one application of the chemical at field-rate. The Cole County, MO silt loam showed larger changes in the microbial community with application of treatments. For the Boone County, MO silty clay loam, Activator 90, Agri-Dex and bentazon treatments increased microbial biomass determined by PLFA; Thrust decreased PLFA markers, bacteria to fungi ratio; and Agri-Dex at both rates decreased monounsaturated fatty acids. Changes in the microbial community due to herbicides or surfactants were minimal in this study of a single application of these chemicals, but could be indicators of potential long-term effects. Long-term studies are needed to determine the changes in the microbial community after several years of annual applications of herbicides and surfactants on a wide array of soil types and management practices.