Sorption may affect the bioavailability and biodegradation of pesticides in soils. The aim of this study was to test the effect of surface sorption on microbial utilization of the herbicide glyphosate as a source of phosphorus, nitrogen, or carbon. We added goethite to a humus soil to manipulate the soil's glyphosate sorption capacity. The addition of glyphosate generally either decreased microbial CO2 production or produced no effect. Additions of glyphosate, in combination with glucose and N, did not change the respiration rate in comparison with the same treatment but without glyphosate. In contrast, glyphosate additions combined with glucose and P decreased microbial growth, whereas the combination with goethite counteracted the negative effect. The different treatments were examined using attenuated total reflectance Fourier transform (ATR-FTIR) spectroscopy; the results suggest that glyphosate was de-carboxylated in the sorbed state. Stimulating microbial growth by the addition of glucose and nitrogen resulted in further oxidation of glyphosate and only phosphate was detectable on the goethite surface after 13 days incubation. Our results show that sorbed glyphosate is microbially degradable, and it retards microbial activity. This study emphasizes the importance of combining quantitative measurements with a molecular-level examination, to better understand biogeochemical processes.