A laboratory experiment was completed to determine the effect of the herbicides Alister Grande 190 OD, Fuego 500 SC and Lumax 537.5 SE on counts of actinomycetes as well as the activity of enzymes and their resistance to herbicides. Sandy loam was mixed with appropriate doses of the herbicides, such as: 0 - the control, 1 - technological dose and doses 20-, 40-, 80- and 160-fold higher than recommended. On day 20, 40, 80 and 160, counts of actinomycetes and activity of urease, acid phosphatase and alkaline phosphatase were determined. For 160 days, soil was incubated at 25°C and its moisture content was maintained on a constant level equal 50% of water capillary capacity. On days 20 and 80 of the experiment, the ecophysiological (EP) and colony development (CD) indices were computed. Additionally, the resistance (RS) of enzymes to the herbicides was assessed on day 20 and their resilience index (RL) was determined on day 160. It has been found out that soil contamination with herbicides contributed to elevated counts of actinomycetes. The highest number of these microorganisms was observed in soil with Lumax 537.5 SE, and the lowest one appeared in soil with Alister Grande 190 OD. The CD for actinomycetes was the highest in treatments with Fuego 500 SC and the highest EP was determined in soil with Alister Grande 190 OD. Application of the herbicides in doses from 20- to 160-fold higher than recommended by the manufacturer significantly increased the activity of acid and alkaline phosphatases. With respect to the activity of urease, the herbicides produced variable effects. The strongest inhibitory effect on the activity of urease was produced by Fuego 500 SC, which reduced the activity of this enzyme by 13.39% when added to soil in a dose exceeding by 160-fold the recommended rate. The RS of the enzymes to the herbicides ranged from 0.461 to 0.955. Urease was the most tolerant to soil contamination with the herbicides.