Abstract A short-term mesocosm experiment was conducted to ascertain the impact of tebuconazole on soil microbial communities. Tebuconazole was applied to soil samples with no previous pesticide history at three rates: 5, 50 and 500 mg kg −1 DW soil. Soil sampling was carried out after 0, 7, 30, 60 and 90 days of incubation to determine tebuconazole concentration and microbial properties with potential as bioindicators of soil health [i.e., basal respiration, substrate-induced respiration, microbial biomass C, enzyme activities (urease, arylsulfatase, β-glucosidase, alkaline phosphatase, dehydrogenase), nitrification rate, and functional community profiling]. Tebuconazole degradation was accurately described by a bi-exponential model (degradation half-lives varied from 9 to 263 days depending on the concentration tested). Basal respiration, substrate-induced respiration, microbial biomass C and enzyme activities were inhibited by tebuconazole. Nitrification rate was also inhibited but only during the first 30 days. Different functional community profiles were observed depending on the tebuconazole concentration used. It was concluded that tebuconazole application decreases soil microbial biomass and activity.