Despite the significant ecological role played by fungi in groundwater, their value as indicators of groundwater pollution is not known, as fungi are notoriously challenging to assess for toxicity because of their varied morphological components, in particular their filamentous structures. Hence they are rarely used in environmental risk assessments and monitoring programs for aquatic systems. A rapid, low cost, 1-step static toxicity test targeting the hyphal components of fungi was developed that subsequently demonstrated the sensitivity of a ubiquitous and dominant groundwater fungal strain to metal contaminants. The strain demonstrates sensitivity to chromium>copper>zinc and relative resistance to arsenic. The introduction of a novel group of groundwater microorganisms-the fungi-for toxicity evaluation will strengthen current risk assessment programs for these ecosystems.