In grazed pastures, soil pH is raised in urine patches, causing dissolution of organic carbon and increased ammonium and nitrate concentrations, with potential effects on the structure and functioning of soil microbial communities. Here we examined the effects of synthetic sheep urine (SU) in a field study on dominant soil bacterial and fungal communities associated with bulk soil and plant roots (rhizoplane), using culture-independent methods and a new approach to investigate the ureolytic community. A differential response of bacteria and fungal communities to SU treatment was observed. The bacterial community showed a clear shift in composition after SU treatment, which was more pronounced in bulk soil than on the rhizoplane. The fungal community did not respond to SU treatment; instead, it was more affected by the time of sampling. Redundancy analysis of data indicated that the variation in the bacterial community was related to change in soil pH, while fungal community was more responsive to dissolution of organic carbon. Like the universal bacterial community, the ureolytic community was influenced by the SU treatment. However, different taxa within the ureolytic bacterial community responded differentially to the treatment. The ureolytic community comprised of members from a range of phylogenetically different taxa and could be used to measure the effect of environmental perturbations on the functional diversity of natural ecosystems.