The entomopathogenic fungus Hirsutella sp., isolated from an infected spider, was found to produce the new cyclotetrapeptide hirsutide (1), cyclo-(L-NMe-Phe-L-Phe-L-NMe-Phe-L-Val), along with the known cytochalasin Q (2), using a cytotoxicity-guided isolation procedure. The structure of 1 was elucidated using one- and two-dimensional NMR experiments, mass spectrometry, and Marfey's method for analyzing the configuration of the amino acids.