In search of a more comprehensive structure-activity relationship (SAR) regarding the inhibitory effect of cytochalasin B (2) on actin polymerization, a virtual docking of 2 onto monomeric actin was conducted. This led to the identification of potentially important functional groups of 2 (i.e., the NH group of the isoindolone core (N-2) and the hydroxy groups at C-7 and C-20) involved in interactions with the residual amino acids of the binding pocket of actin. Chemical modifications of 2 at positions C-7, N-2, and C-20 led to derivatives 3-6, which were analyzed for their bioactivities. Compounds 3-5 exhibited reduced or no cytotoxicity in murine L929 fibroblasts compared to that of 2. Moreover, short- and long-term treatments of human osteosarcoma cells (U-2OS) with 3-6 affected the actin network to a variable extent, partially accompanied by the induction of multinucleation. Derivatives displaying acetylation at C-20 and N-2 were subjected to slow intracellular conversion to highly cytotoxic 2. Together, this study highlights the importance of the hydroxy group at C-7 and the NH function at N-2 for the potency of 2 on the inhibition of actin polymerization.