The content of potassium (K) in soil in relation to the content of other nutrients is a very important factor for plant nutrition. The availability of K for plants is influenced not only by various physical and chemical properties of soil, but also by soil microorganisms. On the other hand, the amount of K and its proportion to other nutrients received influences the amount and the quality of plant root exudates, which can affect the composition and activity of microbial communities. In the current study, the effect of two different K-doses on soil and rhizosphere microorganisms in spring barley was studied in a field experiment. The experiment was performed in a site near Vodnany, South Bohemia, on a cambisol soil type. Two levels of K were applied - 80 kg (which represents an usual dose) and 200 kg K/ha per year, in the form of 60 % KCl. To investigate the reaction of microorganisms, soil and plant, the samples were repeatedly taken and the content of nutrients, physiological groups of microorganisms, microbial biomass and nitrogenase activity were determined. A higher dose of K-fertilization resulted in a higher level of the available K in soil and in a higher K-uptake by barley plants. This was reflected in the elemental composition of the above-ground plant biomass containing more K, N and also P in comparison with that from a less fertilized plot. The amount of microorganisms of various physiological groups increased in the expected sequence: soil between the rows of plants - soil in the rows - rhizosphere soil, without an unambiguous relation to the K-dose. The microbial biomass significantly decreased as influenced with higher K-dose. There were, however, no significant differences between the soil taken in the rows and that taken between the rows of plants. This shows a direct negative influence of K on soil microbial biomass in given soils. The amount of N2-fixers insignificantly increased in the soil taken in the rows as well as in the rhizosphere soil from the plot with a higher K input. The opposite effect of K on nitrogenase activity was observed: soil (taken in the rows of plants) fertilized with more K had a much lower nitrogenase activity than that fertilized with a lower K-dose. This negative effect of K was very much pronounced mainly during May and June, that is during the period of the intensive growth of plants. The effect of a higher K-fertilization on some soil microbial parameters, e.g. repression of nitrogenase activity, decrease of microbial biomass and the changes in the amount of microorganisms of various physiological groups indicate that K can influence soil microorganisms directly and/or indirectly through plants.