The effects of different concentrations of synthetic sheep urine and plant species on ammonia-oxidizing bacterial (AOB) communities in an upland grassland soil were investigated using a microcosm approach. Plant species characteristic of unimproved and improved agricultural pastures (Agrostis capillaris and Lolium perenne, respectively) were planted in soil microcosms, and different levels of synthetic sheep urine were applied, with harvests 10 and 50 days following urine application. Shifts in the community structure of the AOB were investigated using terminal restriction fragment length polymorphism of amoA amplicons. Species richness and diversity were significantly altered by synthetic sheep urine addition and time depending on plant species type. Principal coordinate analysis revealed that AOB community structure was largely dependent on interactions between sheep urine deposition, plant species, and time after urine application, while significant changes in AOB structure were also revealed by similarity percentage analysis. The results of this study suggested that high levels of sheep urine, combined with floristic changes that are characteristic of agricultural intensification, can contribute to temporal and spatial changes in the structure of key bacterial communities in upland grassland soil. Changes in AOB community structure could potentially affect important soil processes, such as nitrification, with subsequent implications for nutrient cycling in agricultural systems.