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The ingestion of vegetables grown in soils or in cultivation substrate contaminated with heavy metals (HMs) and irrigated with wastewater is a potential problem for human health and food quality. The increasing disappearance of fertile soils has led to an increase in the practice of soil-less cultivation and the use of growing substrates, but the choice of the right substrate and its sustainable management is essential to ensure the production of quality and safe vegetables for all while minimizing the impact on the environment and human health. The present study measures the combined effects of different HMs (V, Ni, Cd, Pb, Cu, Cr) on microbial biomass, respiration, and enzyme activities (EAs) in an artificially contaminated commercial growing substrate. The concentrations of HMs were estimated by Atomic Absorption Spectroscopy; enzyme activities via spectrophotometric assays; respiration via CO2 evolution; and microbial biomass C via the fumigation extraction method. The results showed a reduction in both respiration and all enzyme activities. The reduction in EAs highlighted a notable influence on microorganism-mediated C, N, S, and P cycles, strongly reducing substrate health. Microbial biomass did not show significant differences, but the increase in the metabolic quotient highlighted how the toxicity of HMs reduces the energy use efficiency of microbial metabolic processes. [ABSTRACT FROM AUTHOR]