The most modern molecular approaches fail to link structure of soil microbial community with its functions. We used classical physiological approaches based on (i) microbial growth kinetics and (ii) microbial affinity to the substrate to show the shift in functional properties of the soil microbial community after amendments with substrates of contrasting availability. Kinetic parameters of substrate-induced respiration and substrate availability in soil were monitored during the growth of maize, and decomposition of glucose or maize straw. Input of small amounts of easily available substrates (i.e. glucose or root exudates) significantly increased the specific growth rates of soil microorganisms by up to 13 and 20%, respectively. This increase, showing the shift from K to r strategies, was confirmed by a 50% decrease in the affinity of microorganisms to the easily available substrates. In contrast, maize straw lowered specific growth rates by 16–30% and increased the affinity of microorganisms to the substrate by 23–131% compared with untreated soil. After maize straw addition, the shift of the microbial community to K strategy was accompanied by an increase in the fraction of microbial biomass responding to glucose addition by immediate growth. The generation time of this fraction was 1.8 to 2.8 hours, which was 100 to 1000 times faster than that of the whole microbial community. Easily available carbon (C) in soil amended with maize straw comprised only half of that extracted by 0.05 m K2SO4. Therefore, we conclude that C extracted by 0.05 m K2SO4 from the soil with maize straw residues had a lower availability for microorganisms as compared with glucose. Adding N to the soil prolonged the intensive mineralization period of plant residues, decreased specific microbial growth rates and increased the amount of easily available C in the soil. This indicates a more efficient use of maize residues by K-selected microorganisms after the removal of N limitation. Combination of two complementary physiological approaches based on microbial growth kinetics and substrate affinity showed contrasting effects of easily and less available substrates on the shift of growth strategies (r vs. K) of the whole microbial community. These approaches are also suitable for estimation of microbial availability of indigenous C in the soil.