Field experiments were conducted at Ashland Bottoms in northeastern Kansas and at Hays in western Kansas in 2001, 2002, and 2003 to determine the response of soil microbial and nematode communities to different herbicides and tillage practices under a glyphosate-resistant cropping system. Conventional herbicide treatments were a tank mixture of cloransulam plus S-metolachlor plus sulfentrazone for soybean and a commercially available mixture of acetochlor and atrazine for corn. Glyphosate was applied at 1.12 kg ai ha−1 when weeds were 10 or 20 cm tall in both corn and soybean. Soil samples were collected monthly at Ashland Bottoms during the growing period for soil microbial biomass (SMB) carbon determination. In addition, substrate-induced respiration (SIR) and BIOLOG substrate utilization were determined at the end of the growing season each year at Ashland Bottoms, and nematode populations were determined at the beginning and the end of the growing season at both sites. Direct effects of glyphosate rates on soil microbial and nematode communities were also studied in a controlled environment. Values for SMB carbon, SIR, and BIOLOG substrate utilization were not altered by glyphosate. Nematode community response to the glyphosate treatment was similar under both conventional tillage and no-till environments. Total nematode densities were similar with the glyphosate and conventional herbicide treatments. SMB carbon and BIOLOG substrate utilization did not differ between tillage treatments. Nematode densities were greater under conventional tillage than in the no-till system. This study showed that soil health when glyphosate was applied in a glyphosate-resistant cropping system was similar to that of cropping systems that used conventional herbicides.