This study was carried out in order to determine the enzyme activity of the wood vinegar obtained from the hazelnut shells with the potential of bio-pesticide in agro-ecosystem soil, and its effect on the microfungi and heterotrophic bacteria in total. The study was realized in the production seasons of 2014-2015 and 2015-2016, in the ecological conditions of the province Mus (in Turkey) on winter wheat field and with four repetitions according to Randomized Blocks Experimental Design. The treatments within the scope of the experiment were conducted as the six different doses of wood vinegar at 0.5%, 1.0%, 2.0%, 3.0%, 4.0%, and 5.0% mL, and the control treatment which was only provided with tap water. Repeated Measurement ANOVA was used to determine the effect of wood vinegar at different doses, the production seasons and periods (pre- and post-treatment) on relevant features. As based on the statistical inferences, only the Treatment x Year and Period x Year interaction effects in terms of the number of heterotrophic bacteria (P = 0.000 and P = 0.011); and only the Period x Year interaction effects for the number of microfungi (P = 0.000) were found statistically significant. Therefore, the effect of the treatments made on the number of heterotrophic bacteria varied by the production seasons. In a similar way, the effect of the production season and periods on the number of microfungi also changed as based on the term. While the effect of period (P = 0.000) and the Treatment x Period interaction (P = 0.014) effect were significant for Alkaline Phosphatase activity, Year x Period (P = 0.001) and Treatment x Period interactions (P = 0.000) were found significant as related to Beta Glucosidase activity. In conclusion, it may be stated that the wood vinegar used at different doses with the purpose of protecting plant and/or crop in wheat agro-ecosystem does not have a negative effect on microbial factors determined in the soil, and especially, the treatment of 3% mL has a positive effect on bacteria number and Beta Glucosidase enzyme activity.