The main goal of the study was to determine new metagenomic indicators demonstrating sensitivity to soil fatigue as an effect of long-term agricultural use as well as new metagenomic indicators demonstrating bacterial resistance to human agricultural activity. Thirty-one soil samples (agriculturally exploited soil and wastelands – serving as controls) were taken for the study, in the south-eastern part of Poland. For determination of biodiversity, next generation sequencing of the 16S rRNA metagenomic amplicons were used with the Ion Torrent™ technology. Bacterial sequences were clustered into operational taxonomic units (OTUs) based on a 99% similarity threshold. The correlation matrix was constructed to assess the relationships between bacterial families and genera and the environmental data.In the studied soils, 118 families and 305 genera were identified. Among them, 10 families were recommended as sensitive indicators of soil fatigue: Sphingomonadaceae > Chitinophagaceae > Flavobacteriaceae > Oxalobacteraceae > Acetobacteraceae > Myxococcaceae > Comamonadaceae > Pseudomonadaceae > Burkholderiaceae > Rhodanobacteraceae. Analogically, 8 bacterial genera sensitive to agricultural practices were found: Pelomonas > Ramlibacter > Flavobacterium > Rhizobacter > Steroidobacter > Cellvibrio > Halliangium > Pseudomonas. Their sensitivity was confirmed by a decrease in the number of OTUs in the agricultural soils, in comparison to the wastelands. In contrast, 5 bacterial genera that should be considered as indicators of resistance to agricultural land use were proposed: Nitrosospira > Rhodanobacter > Aquicella > Burkholderia > Mucilaginibacter. Statistical tests demonstrated that the most important factors for the abundances of bacterial families and genera included soil acidity (pH), easily degradable carbon (EDC), total carbon (TC), nitrite nitrogen (N-NO2), magnesium (Mg) and calcium (Ca) content, and soil moisture.