Most studies of agroforestry system biodiversity focus on assessing visible, aboveground biodiversity, largely ignoring soil biodiversity. To fill this gap, a preliminary assessment of soil biodiversity in an agroforestry system was undertaken based on changes in soil enzyme activity. The study was conducted in the village of Maziarnia, Lubelskie Voivodeship, Poland, Europe. Arable fields with spring wheat, mid-field trees and perennial mixed forest were selected for the study. Soil material for physicochemical analyses (pHH2O, pHKCl, sorption properties, total carbon and total nitrogen) and biochemical analyses (activity of acid phosphatase, alkaline phosphatase, urease and dehydrogenases) was collected in the spring and autumn of 2022. The present study showed that the biochemical properties of the soils of the selected study sites varied depending on the type of ecosystem determining habitat conditions. Each ecosystem that makes up the agroforestry system studied is characterised by a distinctive microbiome composition and its own level of enzymatic activity. The obtained results support the thesis that agroforestry systems significantly increase the functional diversity and overall biodiversity of agricultural landscapes. However, a full, objective characterisation of the processes taking place in agroforestry systems requires long-term monitoring.