Display omitted •Analogues of the fungal metabolite, eupenifeldin, were generated via semisynthesis.•29 analogues were generated via 22 separate reactions, including esters, carbonates, sulfonates, and carbamates.•Ten analogues maintained nanomolar potency in cytotoxicity assays.•The succinate analogue increased aqueous solubility over 50 times relative to eupenifeldin. Eupenifeldin (1) is a fungal secondary metabolite possessing bis-tropolone moieties that demonstrates nanomolar cytotoxic activity against a number of cancer cell types. As a potential anticancer lead, this meroterpenoid was used to access 29 semisynthetic analogues via functionalization of the reactive hydroxy groups of the bis-tropolones. A series of ester (2–6), carbonate (7–8), sulfonate (9–16), carbamate (17–20), and ether (21–30) analogues of 1 were generated via 22 reactions. Most of these compounds were disubstituted, produced via functionalization of both of the tropolonic hydroxy moieties, although three mono-functionalized analogues (6, 8, and 24) and one tri-functionalized analogue (3) were also obtained. The cytotoxic activities of 1–30 were evaluated against human melanoma and ovarian cancer cell lines (i.e., MDA-MB-435 and OVCAR3, respectively). Ester and carbonate analogues of 1 (i.e., 2–8) maintained cytotoxicity at the nanomolar level, and the greatest improvement in aqueous solubility came from the monosuccinate analogue (6), which was acylated on the secondary hydroxy at the 11 position.