Soil is a pivotal component of the environment and the maintenance of soil quality is critical for ensuring the sustainability of the environment and the biosphere. The use of fertilizers has increased at a faster rate than global food production. Modern agriculture is dependent on phosphorus derived from phosphate rock, which is a non-renewable resource. It is needed to improve P acquisition efficiency for both low input and intensive agroecosystems. However, few studies have focused on cereal-legume interactions with regard to soil P. The objective of this work was to study the effects of grass-legume mixtures and phosphorus fertilization on soil biochemical and microbiological properties. A field experiment including mono-cropping and intercropping of alfalfa and orchard grass at different densities and at two levels of phosphorus fertilization. Soil microbial biomass and the relative abundance of saprophytic fungi and actinobacteria were the microbial parameters most affected by seasonality. The structure of the soil microbial community also changed over years and seasons. Soil basal respiration potential rates, nitrogen mineralization rates, and activities glucosidase and phosphatase were higher in spring and the arylsulphatase activity in summer than in the rest of seasons. The type of plant cover affected both the soil microbial and biochemical parameters. Soil microbial biomass and the relative abundance of mycorrhizal fungi increased with the predominance of orchard grass, while those of saprophytic fungi, actinobacteria and Gram positive and Gram negative bacteria did it with the predominance of alfalfa. In relation to the structure of the soil microbial community, monocrops of orchard grass were very different to the rest of plant cover treatments. Nitrogen mineralization rates were higher in those plant cover treatments with predominance of alfalfa. The soils with high phosphorus fertilization had consistently the lowest levels of arylsulfatase activity.