Livestock slurry containing antibiotics is a source of contamination of agricultural soils, with possible effects on soil function and micro-organisms. Extracellular oxido-reductases and hydrolases from the fungus T. versicolor and fungal growth were monitored in liquid cultures in the presence of tetracycline, lincomycine, sulfadiazine and ciprofloxacin for 10 days, in order to assess the suitability of these enzymes as biomarkers. Among the conditions of treatment, statistical analysis demonstrated an increase in manganese-dependent peroxidase after exposure to sulfadiazine at 1 mg/L when compared with the control. Acid phosphatase activity was decreased by lincomycine at 1 or 10 mg/L. Conversely, β-glucosidase activity increased in the presence of this antibiotic at 10 mg/L. In Terrestrial Model Ecosystems spiked with contaminated pig slurry, lincomycine at the concentration of 8 or 80 μg/kg dry soil, and ciprofloxacin at 250 ng/kg dry soil decreased the activity of soil dehydrogenase, when compared with a green slurry treatment, over 28-day incubations. Laccase activity was similarly decreased in the presence of the highest concentration of antibiotics. We determined bacterial and fungal biomasses using Q-PCR. Bacterial biomass was increased in the presence of lincomycine at 80 μg/kg whatever the time of exposure, and to a lesser extent in the presence of ciprofloxacin at 250 ng/kg, but only at day 28. In contrast, both antibiotics, whatever their concentrations, did not modify fungal biomass in soil. In conclusion, we were unable to demonstrate important effects of antibiotics at concentrations found in the agricultural environment.