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Soil bacteria are major players in driving and regulating ecosystem processes. Thus, the identification of factors shaping the diversity and structure of these communities is crucial for understanding bacterial-mediated processes such as nutrient transformation and cycling. As most studies only target the entire soil bacterial community, the response of active community members to environmental changes is still poorly understood. The objective of this study was to investigate the effect of fertilizer application and sampling time on structure and diversity of potentially active (RNA-based) and the entire (DNA-based) bacterial communities in German grassland soils. Analysis of more than 2.3 million 16S rRNA transcripts and gene sequences derived from amplicon-based sequencing of 16S rRNA genes revealed that fertilizer application and sampling time significantly altered the diversity and composition of entire and active bacterial communities. Although the composition of both the entire and the active bacterial community was correlated with environmental factors such as pH or C/N ratio, the active community showed a higher sensitivity to environmental changes than the entire community. In addition, functional analyses were performed based on predictions derived from 16S rRNA data. Genes encoding the uptake of nitrate/nitrite, nitrification, and denitrification were significantly more abundant in fertilized plots compared to non-fertilized plots. Hence, this study provided novel insights into changes in dynamics and functions of soil bacterial communities as response to season and fertilizer application.