Ecotoxicological research often predominantly relies 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 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 (total Cu content of 26–5,500 mg kg-1) on a chernozem (Mollisol) agricultural field. Earthworm survival in the reproduction bioassay and the average well-color development in the MicroRespTM technique were unreliable predictors of Cu toxicity. In contrast, the number of juveniles in the reproduction bioassay, earthworm avoidance behavior, and basal and substrate-induced respiration were sensitive indicators of Cu toxicity. Total soil Cu predicted earthworm and plant responses as effectively as 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 at 25% (EC25) and 50% (EC50) of total soil Cu for earthworms were 480 and 1005 mg kg-1, respectively, compared to 4,570 and 7,797 mg kg-1, for microbiological responses. Similarly, the EC25 and EC50 of 0.01 M CaCl2-extractable Cu were 14 and 46 μg L-1 for earthworms, though 0.01 M CaCl2-extractable Cu did not predict microbial toxicity well. Overall, earthworms were more sensitive to Cu than microorganisms. This study is among the few that estimate Cu toxicity thresholds in real-world contaminated soils, rather than artificially spiked ones.