ABSTRACT In this study, 28 strains of filamentous fungi were isolated from soil that were historically contaminated with polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs; northern France). These fungal isolates were selected for their ability to use hexadecane as the sole carbon source and for hydrophobic molecule behavior. Six strains produced significant dry biomass (> 25 mg) on hexadecane: Paecilomyces carneus, Penicillium brasilianum, Absidia glauca, Lecanicillium lecanii, Mortierella minutissima, and Bionectria ochroleuca. In a second experiment, P. brasilianum and F. solani were tested for their ability to dissipate 17 PCDD/F congeners in non-sterile contaminated soil microcosms using olive oil as an unconventional carbon source. After two months of incubation, P. brasilianum significantly reduced the soils’ toxicity equivalency (TEQ) by 35%. The F. solani treatment led to a significant reduction (approximately 25%) in PCDD/F concentrations. Statistical agglomerative hierarchical clustering analysis indicated that using cardboard as a growing medium and bulking agent is more efficient than using poplar wood. As fungal dioxin dissipation can involve the production of reactive oxygen species or fungal P450-like enzymes, the potential role of these mechanisms deserve further study. However, the proposed approach could allow the development of cost-effective and eco-friendly (low technology) techniques to maintain the integrity of soils.