Pathogens from nonpoint sources are the leading cause of water quality impairments in US surface waters. This study assessed the capacity of basidiomycetous fungal mycelium on cellulosic substrates to remove Escherichia coli from synthetic stormwater under unsaturated vertical-flow conditions. The mycelium of Stropharia rugoso-annulata was tested in mycofiltration columns consisting of 18.6 L containers with mycelium grown on either wood chips or a mixture of wood chips and straw. S. rugoso-annulata mycofiltration columns were loaded with water spiked with 600–900 cfu/100 mL of E. coli at low (0.5 L/min; 0.57 m/d) and high (2.2 L/min; 2.5 m/d) hydraulic loading. Influent and effluent were monitored for thermotolerant coliform and E. coli using the Coliscan membrane filter chromogenic method. Alder wood chips infused with S. rugoso-annulata mycelium yielded a removal rate of around 20% relative to control filters. Wood chip and straw media appeared less effective with substantial net export of bacteria from both mycelium-infused and un-inoculated control media. The un-inoculated control media used in this study commonly exported high concentrations of thermotolerant coliform bacteria. On wood chip-based media, the presence of actively growing mycelium reduced the thermotolerant coliform exports by >90% relative to the control media. The study highlights the limitations of using thermotolerant coliform to assess pathogen removal in cellulose rich ecotechnologies like mycofiltration.