Increasing nutrient inputs into terrestrial ecosystems affect not only plant communities but also associated soil microbial communities. Studies carried out in predominantly unmanaged ecosystems have found that increasing nitrogen (N) inputs generally decrease soil microbial biomass; less is known about long-term impacts in managed systems such as agroecosystems. The objective of this paper was to analyze the responses of soil microorganisms to mineral fertilizer using data from long-term fertilization trials in cropping systems. A meta-analysis based on 107 datasets from 64 long-term trials from around the world revealed that mineral fertilizer application led to a 15.1% increase in the microbial biomass (Cmic) above levels in unfertilized control treatments. Mineral fertilization also increased soil organic carbon (Corg) content and our results suggest that Corg is a major factor contributing to the overall increase in Cmic with mineral fertilization. The magnitude of the effect of fertilization on Cmic was pH dependent. While fertilization tended to reduce Cmic in soils with a pH below 5 in the fertilized treatment, it had a significantly positive effect at higher soil pH values. Duration of the trial also affected the response of Cmic to fertilization, with increases in Cmic most pronounced in studies with a duration of at least 20 years. The input of N per se does not seem to negatively affect Cmic in cropping systems. The application of urea and ammonia fertilizers, however, can temporarily increase pH, osmotic potential and ammonia concentrations to levels inhibitory to microbial communities. Even though impacts of fertilizers are spatially limited, they may strongly affect soil microbial biomass and community composition in the short term. Long-term repeated mineral N applications may alter microbial community composition even when pH changes are small. How specific microbial groups respond to repeated applications of mineral fertilizers, however, varies considerably and seems to depend on environmental and crop management related factors.