The data nitrogen gene data, soil biodiversity indices and microbial community composition, for three soil depths (0-15, 15-30 and 30-60 cm) from a three-cut silage plot trial located at three grassland sites within the UK collected between April 2016 and October 2016. The sites were Rothamsted Research at North Wyke in Devon, Bangor University at Henfaes Research Station in North Wales and Easter Bush in Scotland. At each site measurements were taken from 16 plots, organised within a randomised complete block design where 4 plots did not receive fertilizers (controls), 4 plots received urea only, 4 plots received urea and urea-inhibitors, and 4 plots received ammonium-nitrate (Nitram). Fertiliser was applied three times and three cuts were performed, all parameters measured were following a fertiliser application. Samples were taken before the fertilizer additions, at peak growth and before the last silage cut. Soil chemical parameters were: soil nitrate, ammonium, dissolved organic carbon and nitrogen, amino acids and peptides, soil organic matter content as loss-on-ignition, pH, sodium, potassium, calcium, magnesium, permanganate oxdisable carbon citric acid extractable phosphorous, Olsen-P and total carbon, nitrogen and phosphorus. Soil biological measure were: microbial biomass carbon and nitrogen. Soil physico-chemical parameters were measured on the same samples and are available in a related dataset measured on the same soil samples and are presented in a separate dataset in the project data series (https://catalogue.ceh.ac.uk/id/7a87dde4-b54e-49b0-8751-1d59e8aebb90). Measurements were undertaken by members of staff from the Centre of Ecology & Hydrology (Bangor, Edinburgh, Lancaster, Wallingford), Bangor University, School of Environment, Natural Resources & Geography and Rothamsted Research, Sustainable Agricultural Sciences, North Wyke. Data was collected for the Newton Fund project 'UK-China Virtual Joint Centre for Improved Nitrogen Agronomy'. Funded by Biotechnology and Biological Sciences Research Council (BBSRC) and NERC - Ref BB/N013468/1