To preserve species-rich grasslands, management practices such as mowing are often required. Mowing is known to promote aboveground conditions that help to maintain plant species richness, but whether belowground effects are important as well is not known. We hypothesized that if mowing decreases belowground carbon transfer by reducing root mass, this will reduce the abundance and activity of soil decomposers and lead to diminished nutrient availability in soil. In grasslands, this would provide a means to mitigate the negative effects of nitrogen enrichment on plant species richness. We established experimental plots on grassland with one-third of plots growing untouched, one-third mowed once a summer, and one-third mowed twice a summer for three growing seasons. Root and soil attributes were measured at each plot 1 month after each mowing. Mowing decreased root mass but had few effects on belowground biota and soil NH4-N, NO3-N, and PO4-P concentrations. Mowing did not affect root N concentrations. Our results show that despite reducing root mass, mowing may have few effects on those soil biota that control nutrient supply and, as a result, have no clear-cut effects on nutrient availability in soil. This suggests that the effects of mowing on soil decomposers and soil nutrient availability may not provide an effective means to mitigate N enrichment and enhance plant species richness in grasslands in a timescale of a few years. Whether such effects could, however, be achieved with longer lasting mowing remains open.