Small molecules that target the actin cytoskeleton have long been recognized as valuable molecular probes and pharmaceutical agents. We have investigated the cellular targets of iriomoteolide-3a and a collection of related macrolide analogues. A new approach for their synthesis has enabled scaffold-diversification and solved the supply problem. Structure-activity relationships suggest that actin is one of iriomoteolides’ primary cellular targets – according to their inhibition of cell migration, induction of morphological changes, reversible cytoplasmic retraction and reduction of F-actin fibers in a time and dose dependent manner. These results showcase iriomoteolides as novel and easily tunable chemical probes for the in vitro study of actin dynamics in the context of cell motility processes including cell invasion and division.