A Phormidium spp. collection from Key West, Florida, afforded caylobolide B (1), an analogue of the known macrolactone caylobolide A, previously isolated from a Lyngbya majuscula collection from the Bahamas. The planar structure of 1 was determined using NMR and MS experiments. The relative configuration for subunits C7-C9 and C25-C29 was assigned using Kishi's Universal NMR Database. Caylobolide B (1) displayed cytotoxic activity against HT29 colorectal adenocarcinoma and HeLa cervical carcinoma cells with IC(50) values of 4.5 and 12.2 μM, respectively.