The structure of emestrin (1), a macrocyclic epidithiodioxopiperazine derivative isolated from the mycelial acetone extract of the thermotolerant fungus Emericella striata (strain 80-NE-22, collected in Nepal), was elucidated using ¹H and ¹³C nuclear magnetic resonance (NMR) spectra and X-ray crystallography of its methanol solvate. Its absolute configuration was determined via circular dichroism (CD) spectroscopy by comparison with known epidithiodioxopiperazines (acetylaranotin and gliotoxin), confirming the 3R,11aR configuration. Emestrin (1) has a molecular formula C₂₇H₂₂N₂O₁₀S₂ (established by field-desorption mass spectrometry (molecular ion at m/z 598) and elemental analysis), with physical properties including a melting point of 233-236 °C (decomposition), [α]ᵈ +184° (c 0.23, CHCl₃), UV absorption maxima (MeOH) at 230, 262, and 278 nm, and IR absorption maxima (KBr) at 3400, 1710, 1680, 1660, and 1610 cm⁻¹. X-ray crystallographic analysis of the methanol solvate (monoclinic, space group, a = 15.256(5) Å, b = 7.788(3) Å, c = 12.203(3) Å, β = 98.16(2)°, Z = 2, Dₓ = 1.53 g cm⁻³) confirmed the structure and relative configuration. Biogenetically derived from two phenylalanine molecules and a benzoate, emestrin exhibits strong antifungal activity. Subsequent to this work, the mycotoxin EQ-1 (isolated from Emericella quadrilineata, E. acristata, and E. parvathecia) was identified as emestrin.