In model laboratory and field conditions, the influence of pollution by antibiotics (benzylpenicillin, ampicillin, streptomycin, oxytetracycline, tylosin, pharmasin, tromexin, aliseryl, and nystatin) on the biological properties of ordinary chernozem was examined in concentrations of 1–1000 mg/kg. A decrease in the majority of the basic biological parameters (ammonifying bacteria, amylolytic bacteria, micromycetes, bacteria of the genus Azotobacter, activity of catalase, dehydrogenase, invertase, phosphatase, peroxidase, polyphenol oxidase) of chernozem occurs when the concentration of antibiotics is 100 mg/kg of soil. In most cases, there was a direct relationship between the content of antibiotics in the soil and the scale of a decrease in the studied parameters. The degree of the influence of antibiotics was determined by their nature, concentration, and time of exposure. Antibacterial antibiotics had more negative impact on the studied indicators than fungicidal ones. By the degree of inhibiting the biological properties of chernozem, antibiotics formed the following sequence: ampicillin > benzylpenicillin ≥ streptomycin ≥ oxytetracycline > tylosin ≥ pharmasin > nystatin > tromexin > aliseryl. Among the examined biological indicators, the number of ammonifying bacteria and the activity of dehydrogenases were reacting most sensitive to a soil pollution by antibiotics. From the investigated enzyme activities, the catalase shows a low response and was no suitable indicator for an antibiotic pollution. The abundance of bacteria of the genus Azotobacter in case of pollution by antibiotics was not informative. The degree of a decrease in biological indicators was more pronounced in laboratory conditions than in the field ones. The rate of the biological activity recovery of chernozem after pollution in the field was 2 times higher. According to the degree of resistance to antibiotics, the investigated microorganisms of chernozem formed the following sequence: bacteria of the genus Azotobacter > micromycetes > amylolytic bacteria > ammonifying bacteria. Enzymes formed the following sequence: peroxidase ≥ polyphenol oxidase > catalase > dehydrogenase > invertase ≥ phosphatase. Antibiotics had prolonged influence on the biological properties of ordinary chernozem. The examined parameters were observed not to be recovered to control values even on the 120th day after the pollution.