The soil of Slovak Republic is severely contaminated with heavy metals, creating hazards to soil health. In order to assess the current status with the prospect of selecting the appropriate treatment methods and land use, this investigation aimed to determine a panel of complementary and ecologically relevant biomarkers that reflect adverse biological responses towards terrestrial pollutants. To attain this objective, the concentration of reduced glutathione and enzymes of glutathione antioxidant system were assessed in clitellate earthworm, Aporrectodea caliginosa sampled from selected sites of eastern Slovakia along with the pH and total metal concentration (As, Cd, Pb, Cr, Hg, Mn, Fe, Co, Ni, Cu, Zn) of soils. Positive, significant (p < 0.05) induction of glutathione peroxidase, glutathione reductase, and glutathione-S-transferase activities and depletion of reduced glutathione level (negative correlation) were associated with the increased soil metal concentrations. Metal interference was found in the detoxification process and antioxidant defense mechanism does not efficiently counteract the oxidative stress induced by chronic metal exposure. The tested biomarkers confirmed sensitive and affective response to the pollution of soil contaminants, in this case metals. This has a potential use in ecotoxicological field monitoring. The proposed principal component analysis is a multivariate model of data analysis that represents a cost-effective approach to differentiate metalliferous soils of eastern Slovakia with different health status. © 2017, Springer International Publishing AG, part of Springer Nature.