Four new compounds (2–5) structurally related to the microtubule-stabilizing agent (−)-zampanolide (1) have been isolated from the Tongan marine sponge Cacospongia mycofijiensis. Three of these new structures, zampanolides B–D (2–4), exhibit nanomolar cytotoxicity toward the HL-60 cell line, are antimitotic, and induce in vitrotubulin polymerization at levels comparable to 1. Zampanolide E (5), saturated at C-8/C-9, was significantly less potent and does not stabilize purified tubulin, even at 10-fold higher concentrations. The structural differences across these compounds reveal a plasticity of the zampanolide pharmacophore. While unsaturation is required at Δ8, the configuration of this alkene and those of Δ4and Δ4′have little effect on tubulin polymerization. The first natural co-occurrence of 1and (−)-dactylolide (6) from the same sponge extract is also noted.