Digestate from biogas production contains a significant amount in organic matter and N, whereas fly ash from biomass combustion is rich in P and other nutrients. The use of both, as suitable fertilizers, has been shown to improve crop yields and soil properties. A Chernozem soil was used in a pot experiment in which wheat was grown to evaluate the impact of digestate (organic amendment) and fly ash (inorganic amendment) application on soil microbial activity, biomass and community structure. Soil samples were collected after two time-term treatments (30 and 60 days). Digestate application significantly improved soil chemical variables, microbial activity, the substrate average well colour development (SAWCD) and physiological diversity. Bacterial, actinobacterial, and fungal communities were positively affected by digestate addition, as well as microbial biomass. These effects were evident after a short period (30 days), but were more significant after long-term treatment (60 days). However, fly ash did not provoke significant changes in soil microbial activities, qPCR or PLFA at any time of treatment. Nevertheless, with this amendment, some chemical variables were positively affected after 60 days of treatment and the SAWCD revealed different patterns in the use of C sources compared with un-amended and digestate-amended soil at both treatment times. These results suggest that the chemical properties present in digestate may determine its use as a suitable organic amendment because it improves soil health and quality, whereas fly ash did not have a relevant impact on soil microbial activities, qPCR, or PLFA.