The total synthesis of ratjadone enables a thorough investigation of its mode of action and of structure–activity relationships. In this highly convergent approach, three subunits are joined together by a Wittig olefination and a selective Heck reaction as the pivotal steps. Diastereomeric ratjadones were synthesized by using enantiomeric subunits. They display a range of biological activities that can be correlated to their overall conformational preferences. The overlay of two diastereomers shows that the configuration at C10 induces a helix-like conformation.