Ecotoxicological research often relies predominantly on artificially contaminated soils, with studies on real-world contaminated soils remaining scarce. This study focuses on the Kargaly site in the Orenburg region of Russia, a rare instance of monometallic soil pollution by copper (Cu). The similarity of other elements in the soil samples to background levels highlights the uniqueness of this monometallic contamination. We established Cu toxicity thresholds for soil microorganisms and earthworms using soils collected along a Cu toxicity gradient in a chernozem (Mollisol) agricultural field. The total soil Cu predicted earthworm responses as effectively as did 0.01 M CaCl2-extractable Cu. While total soil Cu strongly predicted microbiological responses, 0.01 M CaCl2-extractable Cu was a poor predictor for microorganisms. The effective concentrations of total soil Cu for earthworms at 25% (EC25) and 50% (EC50) were 480 and 1005 mg kg-1, respectively, compared to 4,570 and 7,797 mg kg-1 for microorganisms, respectively. Similarly, the EC25 and EC50 of 0.01 M CaCl2-extractable Cu were 14 and 46 µg L-1 for earthworms, respectively, although 0.01 M CaCl2-extractable Cu did not predict microbial toxicity well. Overall, earthworms were more sensitive to Cu than were microorganisms. This study is among the few that estimate Cu toxicity thresholds in real-world contaminated soils rather than artificially spiked soils.