Soil-dwelling microorganisms play an important role in the environment by decomposing organic matter, degrading toxic compounds and participating in the nutrient cycle. The microbiological properties of soil are determined mainly by the soil pH, granulometric composition, temperature and organic carbon content. In agricultural soils, these parameters are modified by agronomic operations, in particular fertilization. Soil enzymes participate in nutrient cycling and they are regarded as sensitive indicators of microbial activity and changes in the soil environment. The aim of the present study was to determine whether PAH content in soil is associated with the microbial activity and biochemical properties of soil during the growing season of spring barley treated with manure and mineral fertilizers. Soil samples for analysis were collected on four dates in 2015 from a long-term field experiment established in 1986 in Bałcyny near Ostróda (Poland). The total content of PAHs was lowest in August (194.8 µg kg−1) and highest in May (484.6 µg kg−1), whereas the concentrations of heavier weight PAHs was highest in September (158.3 µg kg−1). The study demonstrated that weather conditions and microbial activity induced considerable seasonal variations in PAHs content. Manure increased the content of organic carbon and total nitrogen, the abundance of organotrophic, ammonifying and nitrogen-fixing bacteria, actinobacteria and fungi and enhanced the activity of soil enzymes, including dehydrogenases, catalase, urease, acid phosphatase and alkaline phosphatase.