We evaluate the use of signature fatty acids and direct hyphal counts as tools to detect and quantify arbuscular mycorrhizal (AM) and saprotrophic fungal (SF) biomass in three Hawaiian soils along a natural soil fertility gradient. Phospholipids16:1omega5c and 18:2omega6,9c were used as an index of AM and saprotrophic fungal biomass, respectively. Both phospholipid analysis and hyphal length indicated that the biomass of AMF was greatest at the highest fertility site, and lowest where phosphorus limits plant growth. Saprotrophic fungal biomass did not vary. Hyphal length counts appeared to under-estimate SF abundance, while the phospholipid AMF:SF ratio was in line with expectations. This study indicates that phospholipids may be a valuable and reliable tool for studying the abundance, distribution, and interactions between AM and saprotrophic fungi in soil.