Fungi impact humans and the environment in many ways, for good and ill. Some fungi support the growth of terrestrial plants or are used in biotechnology, and yet others are established or emerging pathogens. In some cases, the same organism may play different roles depending on the context or the circumstance. A better understanding of the relationship between fungal biochemical composition as related to the fungal growth environment is essential if we are to support or control their activities. Synchrotron FTIR (sFTIR) spectromicroscopy of fungal hyphae is a major new tool for exploring cell composition at a high spatial resolution. Brilliant synchrotron light is essential for this analysis due to the small size of fungal hyphae. sFTIR biochemical characterization of subcellular variation in hyphal composition will allow detailed exploration of fungal responses to experimental treatments and to environmental factors.