Microbial mats are a visible and abundant life form inhabiting the extreme environments in Yellowstone National Park (YNP), WY, USA. Little is known of their role in food webs that exist in the Park's geothermal habitats. Eukaryotic green algae associated with a phototrophic green/purple Zygogonium microbial mat community that inhabits low-temperature regions of acidic (pH ∼ 3.0) thermal springs were found to serve as a food source for stratiomyid (Diptera: Stratiomyidae) larvae. Mercury in spring source water was taken up and concentrated by the mat biomass. Monomethylmercury compounds (MeHg+), while undetectable or near the detection limit (0.025 ng l−1) in the source water of the springs, was present at concentrations of 4–7 ng g−1 dry weight of mat biomass. Detection of MeHg+ in tracheal tissue of larvae grazing the mat suggests that MeHg+ enters this geothermal food web through the phototrophic microbial mat community. The concentration of MeHg+ was two to five times higher in larval tissue than mat biomass indicating MeHg+ biomagnification occurred between primary producer and primary consumer trophic levels. The Zygogonium mat community and stratiomyid larvae may also play a role in the transfer of MeHg+ to species in the food web whose range extends beyond a particular geothermal feature of YNP.