Links between the microbial community structure and soil functions are unclear. The study of these relationships requires the development of highly specific experimental approaches. In this work, the soil microbial community structure and function relationship was evaluated in relation to heat stress in a soil microcosm incubated at 17 °C and 50 °C. We selected a luvisol that included two land uses. Samples were taken from the soil of a long-term (>10 year) arable cropping plot (CC) and a permanent grassland (PG) (>25 years). The soil functions were evaluated by measuring the enzyme activities, including cellulase, N-acetyl-glucosaminidase, β-glucosidase, xylanase and dehydrogenase. The total microbial biomass was assayed by the quantification of the total DNA extracted from the microcosm soils. The abundance of total bacterial and fungal communities and different bacterial taxa were measured by qPCR rRNA genes. For both soil types, heat stress induced changes in the microbial community structure and soil functions. In most cases, the results yielded effects following heat treatment. All of the enzymes were inhibited except xylanase. Heat stress significantly reduced the total microbial biomass and fungal abundance in the soils. The abundance of the total bacterial community was not affected by heat stress. In the two soils, the dominant taxa were Actinobacteria (13–40%) and Bacteroidetes (14–32%), while Planctomycetes and Gammaproteobacteria exhibited lower abundance (0–3%). Changes in the microbial community structure and changes in the functions were correlated; the correlation was positive in the PG soil and negative in the CC soil. The changes in the CC soil structural and functional state were greater than of those observed in PG soil. Our initial hypothesis was confirmed, indeed, grassland soil is more resistant to drastic stress due to its highly abundant and highly diversified microbial community. These results represent a contribution to the understanding of soil microbial community structure and functions relationships.