Soil organic matter is composed of a variety of carbon (C) forms. However, not all forms are equally accessible to soil microorganisms. Deprivation of C inputs will cause changes in the physical and microbial community structures of soils; yet the trajectories of such changes are not clear. We assessed microbial communities using phospholipid fatty acid profiling, metabarcoding, CO2 emissions, and functional gene microarrays in a decade-long C deprivation field experiment. We also assessed changes in a range of soil physicochemical properties, including using X-ray Computed Tomography imaging to assess differences in soil structure. Two sets of soils were deprived of C inputs by removing plant inputs for 10 years and 1 year, respectively. We found a reduction in diversity measures, after 10 years of C deprivation, which was unexpected based on previous research. Fungi appeared to be most impacted, likely due to competition for scarce resources after exhausting the available plant material. This suggestion was supported by evidence of bioindicator taxa in non-vegetated soils that may directly compete with or consume fungi. There was also a reduction in copies of most functional genes after 10 years of C deprivation, though gene copies increased for phytase and some genes involved in decomposing recalcitrant C and methanogenesis. Additionally, soils under C deprivation displayed expected reductions in pH, organic C, nitrogen, and biomass as well as reduced mean pore size, especially in larger pores. However, pore connectivity increased after 10 years of C deprivation contrary to expectations. Our results highlight concurrent collapse of soil structure and biodiversity following long-term C deprivation. Overall, this study shows the negative trajectory of continuous C deprivation and loss of organic matter on a wide range of soil quality indicators and microorganisms.