This study compares the effect of application of digestate, straw, cattle slurry and inorganic fertilizers on crop yield and soil organic matter content. Total organic carbon (C), total organic nitrogen (N), hot water soluble C, microbial biomass C and hydrophobic soil components were evaluated in soil from the field experiment in PragueRuzyně (Orthic Luvisol, clay loam) with winter wheat. All fertilized treatments significantly increased grain yield above the level of non-fertilized control (5.68 t/ha), and the sequence was as follows: digestate (9.88 t/ha) > NPK (9.80 t/ha) > cattle slurry (9.73 t/ha) > digestate + straw (9.35 t/ha). Average organic C content in the soil ranged from 1.668-1.704% and the effect of different fertilization was not significant. The highest increase of microbial biomass C was recorded in digestate + straw (43.2% increase compared to control). Highly significant correlations were found between hydrophobic soil components and hot water soluble C (r = 0.988; P ≥ 0.05) and microbial biomass C (r = 0.964; P ≥ 0.05). Total organic N content ranged from 0.157-0.160% and differences among treatments were insignificant. Fertilization with digestate itself brings an effect in increasing crop yield, but does not improve the level of soil organic matter significantly.