Two soil microbial processes, substrate-induced nitrification (SIN) and substrate-induced respiration (SIR), were measured in the topsoils of 12 Australian field trials that were amended separately with increasing concentrations of ZnSO4 or CuSO4. The median effect concentration (EC50) values for Zn and Cu based on total metal concentrations varied between 107 and 8,298 mg kg−1 for Zn and 108 and 2,155 mg kg−1 Cu among soils. The differences in both Zn and Cu toxicity across the 12 soils were not explained by either the soil solution metal concentrations or CaCl2-extractable metal concentrations, because the variation in the EC50 values was larger than those using total concentrations. Toxicity of Zn and Cu decreased with increasing soil pH for SIN. For Cu, also increasing cation exchange capacity (CEC) and percent clay decreased the toxicity towards SIN. In contrast to SIN, soil pH had no significant effect on toxicity values of SIR. Significant relationships were found between the EC50 values for SIR and background Zn and CEC for Zn, and percent clay and log CEC for Cu. Relationships such as those developed in this study will permit Australian environmental regulation to move from single-value national soil quality guidelines to soil-specific quality guidelines and permit soil-specific risk assessments to be undertaken.