The increased use of sulfate fertilizers to compensate for soil sulphur (S) limitation in agricultural soils may affect soil microbes and micro-fauna involved in S mobilization. Here, columns with podzolic soil material and ryegrass (Lolium perenne) were fertilized with 0, 5, 10 and 20 kg ha-1 (S0/S5/S10/S20) inorganic sulfate-S alongside a full complement of other nutrients. In the S10 and S20 columns, significantly higher amounts of sulfate were present in soil solution. After two grass cuts (14 weeks in total), there was a significant decrease in arylsulfatase activity, bacterial-feeding nematode abundances and mycorrhizal colonization in the S10 and S20 columns compared to the S0. Bacterial, fungal and AM community structures shifted significantly across the treatments. After final harvest, the S10 and S20 columns had significantly higher grass dry matter yield and uptake of S, N, K, Ca and Mg compared to the S0. While the overall bacterial diversity was reduced in the S20 treatment, abundance (asfA) and diversity (ssuD and atsA) of bacterial genes involved in S cycling were not significantly affected by one-time sulfate fertilization. These results indicate that short-term sulfate fertilization benefits to plant growth outweighed the negative feedback from parts of the soil biota. To improve nutrient use efficiencies in a sustainable manner, future studies should consider alternative S fertilizers which may be beneficial to both, the soil biota and plants in the long-term.