The objective of the study was to determine the dynamics of the enzymatic activity in a substrate containing a microbiological inoculum (Effective Microorganisms – EM) intended to improve French marigold growth and flowering. The material used in the study was peat substrate of 5.5–6.0 pH into which plants were planted and then inoculated with different doses of the EM biofertilizer (1 : 10, 1 : 50, 1 : 100). Samples of the substrate on which experimental plants were cultivated were collected during the following three phases: the phase of seedling planting, the phase of vegetative growth and the phase of flowering. The scope of investigations comprised determination of the activity of dehydrogenases, urease and acid phosphatase activity by the spectrophotometric method. In addition, the following plant morphological analyses were per-formed: plant height, shoot number and length, number of buds and inflorescences, and leaf greenness index (SPAD). It was found that the phase of plant development was proved to be the main determinant of the activity of the enzymes under study. The EM biofertilizer had positive effect on the acid phosphatase activity. However, the preparation did not have stimulating effect on the activity of urease or dehydrogenases. The EM biofertilizer concentrated at 1 : 100 and applied into the soil and foliage resulted in a larger number of darker leaves and greater number of inflorescences on the plants.