<jats:title>Abstract</jats:title><jats:p>(−)‐Alternarlactam [(−)‐<jats:bold>1</jats:bold>], a new promising cytotoxin against two human cancer cell lines, was isolated from an endophyte culture and synthesized (along with (+)‐<jats:bold>1</jats:bold>) from readily available starting materials. The absolute configuration, chirality–activity relevance and self‐aggregation of (−)‐<jats:bold>1</jats:bold> were assigned by a combination of synthetic, spectroscopic and computational approaches. The full characterization of the new fungal cytotoxin may provide valuable information in the discovery of new antitumor agents.