Microbial functioning refers to microbial activity because only the active microorganisms drive biogeochemical processes. Despite the importance of active microorganisms, most methods focus on estimating total microbial biomass and fail to evaluate its active fraction. At first, we have described the differences among the active, potentially active, and dormant microbial states in soil and suggested threshold values of parameters for their identification. Secondly, we critically reviewed the ability of a broad range of approaches to estimate and characterize the active and the potentially active microorganisms in soil. Following approaches were evaluated: plate count and microbial cultures; direct microscopy combined with cell staining; ATP, PLFA, DNA and RNA content; microarray analyses; PCR-based approaches; stable isotope probing; soil proteomics, enzymes activity; and various approaches based on respiration and substrate utilization. The “static” approaches, mainly based on the single-stage determination of cell components (ATP, DNA, RNA, and molecular biomarkers), detect well the presence of microorganisms and total biomass, but they fail to evaluate the active part and consequently the functions. In contrast, the dynamic approaches, estimating the changes of these parameters during microbial growth and based on process rates: substrate utilization and product formation, e.g., respiration, help to evaluate active microbial biomass and relate it to specific process rates. Based on a comparison of all approaches for their universality (possibility to analyze active, potentially active and dormant microorganisms), we concluded that 1) direct microscopy with complementary stains, 2) a combination of RNA-based FISH with staining of total microbial biomass, and 3) approaches based on microbial growth were the most advantageous and allowed simultaneous quantitative estimation of active, potentially active, and dormant microorganisms in soil. The active microorganisms compose only about 0.1–2% of the total microbial biomass and very seldom exceed 5% in soils without input of easily available substrates. Nonetheless, the fraction of potentially active microorganisms (ready to start utilization of available substrates within few hours) is much higher, contributing between 10 and 40% (up to 60%) of the total microbial biomass. Therefore, we emphasize the role of potentially active microorganisms with quick response to fluctuating substrate input in soil microhabitats and hotspots. The transition from the potentially active to the active state occurs in minutes to hours, but the shift from dormant to active state takes anywhere from hours to days. Despite very fast activation, the reverse process – fading to the potentially active and dormant stage – requires a much longer period and is very different for individual criteria: ATP, DNA, RNA, enzyme production, respiration rates. This leads to further difficulties in the estimation of the active part of microbial community by methods based on these parameters. Consequently, the standardization, further elaboration, and broad application of approaches focused on the portion of active microorganisms in soil and their functions are urgently needed. We conclude that because active microorganisms are the solely microbial drivers of main biogeochemical processes, analyses of the active and potentially active fractions are necessary in studies focused on soil functions. Display omitted •We critically evaluated literature on active soil microorganisms and revealed.•Microorganisms in active and potentially active state: tiny versus large pool.•Threshold parameters for microbial activity state in soil.•Relevance of active microbial fraction in calculations of process rates.•Combinations of dynamic approaches enable to evaluate active microbial biomass.