Effects of the fungicide propiconazole on soil microorganisms were tested using [3H] leucine incorporation and [14C] acetate in ergosterol incorporation to measure bacterial and fungal growth inhibition, respectively. Growth was compared to basal respiration (BR) and substrate-induced respiration (SIR) in soil microcosms established according to the OECD 217 guideline. Fungal growth was most sensitive with IC50 values remaining around 300 mg kg−1 during 40 days of incubation. SIR was initially less sensitive (IC50 1300 mg kg−1), but IC50 values progressively decreased over time to reach 380 mg kg−1 after 40 days. Bacterial growth was affected at concentrations ≥200 mg kg−1, but exhibited more complex dose-response relationships possibly due to a combination of direct toxicity, bacterial community adaptation, and competitive release from the more severely affected fungi. BR was either stimulated or not affected by propiconazole. Our results indicate that group-specific endpoints targeting microbial growth will improve ecotoxicological assessment of toxicants for environmental risk assessment.