Glyphosate is the world's most widely applied herbicide. Despite its extensive and heavy use in agriculture and forestry, the microbial key players involved in glyphosate breakdown and the impact of this compound on the soil microbiota including fungi is not fully understood. Here, we used microcosm experiments to determine the impact of glyphosate application on the microbial community structure and abundance of four different agricultural soils from the Ammer valley, Germany, with a history of glyphosate application. We identified putative glyphosate degraders that could be active under in-situ conditions. Glyphosate was applied to the soils in a single dose of 15 mg·kg−1, which were incubated in the dark with 60 % water-filled pore space at 20 °C for 56 days. Soil samples were taken on the day of glyphosate addition (day 0) and after 7, 28 and 56 days. Capillary electrophoresis-mass spectrometry was used to quantify glyphosate and its major transformation product aminomethylphosphonic acid. Changes in the microbial community structure and abundance were evaluated using 16S rRNA gene or internal transcribed spacer region amplicon sequencing and qPCR. Our findings demonstrate that glyphosate was rapidly degraded in the four soils, with 60–85 % of the applied glyphosate disappearing within 7 days. However, the observed impact of glyphosate application on the microbial community composition was minimal and, notably, there was no significant time-dependent glyphosate effect at 7 days across all four soils. This suggests that glyphosate degradation in soil might be a concerted effort by a wide microbial network or it might be occurring co-metabolically. In addition, the sorption/desorption dynamics of glyphosate with the soil matrix can heavily influence its bioavailability, further reasoning the subtle effects observed on the microbial community structure.