<jats:title>Abstract</jats:title><jats:p>A new tetrapeptide <jats:sc>D</jats:sc>‐Phe‐<jats:sc>L</jats:sc>‐Val‐<jats:sc>D</jats:sc>‐Val‐<jats:sc>L</jats:sc>‐Tyr (<jats:bold>1</jats:bold>), along with three known diketopiperazines and pseurotin A, were isolated from the culture of <jats:italic>Penicillium canescens</jats:italic>, collected from pollen from beehives, in a screening for new antimicrobial products from unexplored sources. The structure of the tetrapeptide, which exhibits antifungal activity comparable with that of the commercial product benomyl against the soybean phytopathogen <jats:italic>Fusarium virguliforme</jats:italic>, was determined by spectroscopic (2D‐NMR, and MS and MS/MS) and chemical methods, and the sequence was confirmed by comparison with authentic synthetic isomeric peptides.