<jats:p>Three new cycloheximide congeners, 2,3-dehydro-α-<jats:italic>epi</jats:italic>-isocycloheximide (<jats:bold>1</jats:bold>), (<jats:italic>E</jats:italic>)- and (<jats:italic>Z</jats:italic>)-2,3-dehydroanhydrocycloheximides (<jats:bold>2</jats:bold> and <jats:bold>3</jats:bold>), together with three known compounds, anhydroisoheximide (<jats:bold>4</jats:bold>), cycloheximide (<jats:bold>5</jats:bold>), and isocycloheximide (<jats:bold>6</jats:bold>), were obtained from the cultures of <jats:italic>Streptomyces</jats:italic> sp. SC0581. Their structures were elucidated by extensive spectroscopic analysis in combination with theoretical conformational analysis and ECD computations. The photoinduced interconversion between <jats:bold>2</jats:bold> and <jats:bold>3</jats:bold> was observed and verified and the possible reaction path and mechanism were proposed by theoretical computations. The antifungal and cytotoxic activities of <jats:bold>1</jats:bold>–<jats:bold>6</jats:bold> were evaluated and suggested that 2,3-dehydrogenation results in the loss of the activities and supported that the OH-α is important to the activities of cycloheximide congeners.