The use of fungicides bears the risk of many undesirable outcomes that are manifested in, among other things, changes in the structure and activity of microorganisms. This study aimed at determining the effect of a Helicur 250 EW preparation, used to protect crops against fungal diseases, on the microbiological and biochemical activity of soil and on the development of Horderum vulgare L. The fungicide was sprayed on leaves of spring barley in the following doses (per active substance, i.e. tebuconazole, TEB): 0.046, 0.093, 0.139, 1.395, and 2.790 mg TEB plant−1. The following indices were analyzed in the study: index of microorganisms resistance (RS) to the effects of fungicide, microorganisms colony development index (CD), microorganisms ecophysiological diversity index (EP), genetic diversity of bacteria, enzymatic activity, and effect of the fungicide on spring barley development (IFH). The most susceptible to the effects of the fungicide turned out to be fungi. The metagenomic analysis demonstrated that the bacterial community differed in terms of structure and percentage contribution in the soil exposed to the fungicide from the control soil even at the Phylum level. However, Proteobacteria appeared to be the prevailing taxon in both soils. Bacillus arabhattai, B. soli, and B. simplex occurred exclusively in the control soil, whereas Ramlibacter tataounensis, Azospirillum palatum, and Kaistobacter terrae – exclusively in the soil contaminated with the fungicide. Helicur 250 EW suppressed activities of all soil enzymes except for arylsulfatase. In addition, it proved to be a strong inhibitor of spring barley growth and development. Display omitted •Helicur 250 EW contributed to the disturbance of soil homeostasis.•The fungicide changed the genetic diversity of bacteria and enzymatic activity in soil.•Microorganisms, enzymes and spring barley can be used as a bioindicators.