Greenhouse and laboratory experiments were conducted on common waterhemp and soil collected from 131 soybean fields in Missouri that contained late-season common waterhemp escapes. The objectives of these experiments were to determine the effects of soil sterilization on glyphosate-resistant (GR) and -susceptible (GS) common waterhemp survival, to determine the effects of soil sterilization and glyphosate treatment on infection of GR and GS common waterhemp biotypes by Fusarium spp., and to determine the soil microbial abundance and diversity in soils collected from soybean fields with differences in common waterhemp biotypes and herbicide and crop rotation histories. Common waterhemp biotypes were treated with 1.7 kg glyphosate ae ha−1 or left untreated once plants reached approximately 15 cm in height. Common waterhemp survival was visually assessed at 21 d after glyphosate treatment (21 DAT). To determine Fusarium infection frequency, a single intact common waterhemp root was harvested from each treatment at 0, 3, 7, 14, and 21 DAT and surface sterilized, and 10 to 15–mm common waterhemp root sections were plated on Komada culture medium. After 14 d incubation, fungal colonies were selected from colonized roots and maintained on potato dextrose agar medium amended with antibiotics before identification. Speciation of Fusarium isolates was conducted through microscopic examination of fungal characters and confirmed by sequencing and analysis of ribosomal DNA. Soil samples from 131 different collections were subjected to phospholipid fatty acid (PLFA) analysis and were conducted utilizing gas chromatography to determine the soil microbial community abundance and structure. Common waterhemp plants grown in sterile soils had the highest common waterhemp survival, regardless of biotype. After treatment with glyphosate, survival of GS common waterhemp grown in nonsterile soil was only 29% 21 DAT, whereas survival of GS common waterhemp grown in nonsterile soil was only 10%. Similarly, GR common waterhemp survival was reduced from 83 to 61% following treatment with glyphosate when grown in nonsterile compared to sterile soil. Fusarium spp. were recovered from only 12% of the assayed roots (223 treatments with Fusarium out of a total 1,920 treatments). The greatest occurrence of Fusarium root infection in both GR and GS common waterhemp occurred in nonsterile soils following a glyphosate treatment. Few differences in total PLFA were observed in field soil collected from locations with either GR or GS common waterhemp, and regardless of herbicide or crop history. This research supports previous findings that plant species are more sensitive to glyphosate in nonsterile than sterile soils and indicates glyphosate may predispose plants to soil-borne phytopathogens. This research also suggests that continuous use of glyphosate does not significantly affect soil microbial abundance or diversity.