© 2020, Springer-Verlag GmbH Germany, part of Springer Nature. Enhancing the role of the soil microbiota in plant phosphorus (P) and sulfur (S) supply through application of organic fertilizer could reduce dependencies on non-sustainable synthetic fertilizers. To compare the effects of organic/inorganic fertilizers on the soil microbiota, soil columns with Lolium perenne (ryegrass) were set up in a greenhouse and amended with an inorganic fertilizer, cattle slurry (organic), or urea (P- and S-free control). Ryegrass rhizosphere of the slurry treatment had significantly higher abundances of bacterial feeding nematodes, mycorrhizal colonization, cultivable heterotrophic bacteria, phosphonate- and sulfonate-utilizing bacteria, arylsulfatase activity, available P, and Variovorax asfA gene copies compared to the inorganic and urea treatments. Phosphomonoesterase activities, and gene abundances involved in organic P and S transformations (phoD, phoC, Burkholderia, and Polaromonas asfA) were similar in all treatments. Grass dry matter yield and shoot uptake of N, P, and S were significantly higher in the inorganic treatment compared to the urea and slurry treatments. Community compositions differed significantly between the three fertilizer treatments and included the bacterial, alkaline phosphomonoesterase-producing bacterial, fungal, AM fungal, and nematode communities. Bacteriodetes were found in higher relative abundance in the organic treatment, while Acidobacteria were more abundant in the urea and inorganic fertilizer treatments. These community shifts correlated significantly with grass dry matter yield, uptake of N, P, and S, mycorrhizal colonization, enzyme activities, abundances of bacteria, and bacterial feeding nematodes. We concluded that organic fertilization promoted soil microbes and nematodes which have the potential to support sustainable plant growth, provided that the overall nutrient requirements are met.