Aims: This work has examined the effects of a polycyclic aromatic hydrocarbon and selected toxic metals on fungal populations in a soil microcosm. Methods and Results: By using fungal ribosomal intergenic spacer analysis (F-RISA) in combination with real-time PCR quantification, four fungi (D63P2-1, D63C2-1, D21Cu1-1 and D63Pb2-2) with specific primer pairs to each were successfully evaluated for their potential as bioindicators in response to pyrene, copper (Cu) and lead (Pb), supplied singly and in combination. Conclusions: F-RISA coupled with real-time PCR is a useful approach for the identification of microorganisms with potential as bioindicators of organic and toxic metal contamination. Significance and Impact of the Study: These bioindicators could be monitored for their population changes that may indicate pollutant-induced perturbations in a given system.