Several lines of evidence suggest that the agaricoid, non-ectomycorrhizal members of the family Hygrophoraceae (waxcaps) are biotrophic with unusual nitrogen nutrition. However, methods for the axenic culture and lab-based study of these organisms remain to be developed, so our current knowledge is limited to field-based investigations. Addition of nitrogen, lime or organophosphate pesticide at an experimental field site (Sourhope) suppressed fruiting of waxcap basidiocarps. Furthermore, stable isotope natural abundance in basidiocarps were unusually high in ¹⁵N and low in ¹³C, the latter consistent with mycorrhizal nutritional status. Similar patterns were found in waxcap basidiocarps from diverse habitats across four continents. Additional data from ¹⁴C analysis of basidiocarps and ¹³C pulse label experiments suggest that these fungi are not saprotrophs but rather biotrophic endophytes and possibly mycorrhizal. The consistently high but variable δ¹⁵N values (10–20‰) of basidiocarps further indicate that N acquisition or processing differ from other fungi; we suggest that N may be derived from acquisition of N via soil fauna high in the food chain.