Application of N-fertilizers can lose nitrogen to the atmosphere via ammonia (NH3) volatilization and also affect survival of microbial communities in the soil. Here, an agricultural soil was supplemented with urea, ammonium sulfate, potassium nitrate or urea together with the urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) and maintained at 50 and 80% water-filled pore space (WFPS). Ammonia volatilization was monitored daily for 15 days. Abundance of total bacterial and archaeal populations and those of the nitrifier and denitrifier communities were determined by quantitative PCR of their corresponding 16S rRNA, amoA and norB, nosZI and nosZII genes. The highest NH3 fluxes were detected at 50% WFPS after urea application and ammonium had intermediate volatilization potential regarding the control soil. Urea + NBPT reduced ammonia emissions at 50 and 80% WFPS. Addition of any of the fertilizers increased the total abundance of Bacteria and Archaea regardless of the moisture conditions, and urea + NBPT had no effect on soil microbial communities. Ammonium-based fertilizers increased the abundance of the PCR-amplified nitrification genes from soil since the beginning, values that were delayed when urea + NBPT was added. Nitrogen fertilization increased the abundance of denitrifiers, particularly after nitrate application. Urea + NBPT did not affect the total abundance of the denitrification genes.