In the course of our screening program for novel metabolites from tunicate-derived fungi, novel sesquiterpenoids, named JBIR-27 (1) and -28 (2), together with known sporogen-AO1 and phomenone, were isolated from the culture broth of Penicillium sp. SS080624SCf1. The structures of 1 and 2 were determined to be eremophilane analogs on the basis of extensive NMR and MS analyses. Sporogen-AO1, phomenone and 2 showed cytotoxicity against human cervical carcinoma cell line HeLa at IC 50 values of 8.3, 19 and 92 mM, respectively, whereas 1 was inactive at a concentration of 80 mM. Keywords: cytotoxicity; JBIR-27; JBIR-28; Penicillium sp.; terpenoid; tunicate INTRODUCTION Many bioactive substances have been isolated from marine organisms, such as marine microorganisms, phytoplankton, algae, sponges and tunicates. Moreover, marine microorganisms have been studied as the important resources for new biologically active metabolites. 1 Especially, marine-derived fungi are emerging as an attractive source for discovering new bioactive compounds. 2 Indeed, cytotoxic metabolites, such as diketopiperazine alkaloids, 3 trichodermatides 4 and carbonarones, 5 have been isolated from metabolites of marine-derived fungi. Tunicates are a rich source of unique and biologically active metabolites. 1 However, there have been few reports of compounds isolated from tunicate-derived fungi. 6,7 Therefore, we attempted to isolate fungi from a tunicate, Didemnum molle, and obtain secondary metabolites from the fungal culture broths.In the course of chemical screening for novel compounds from the metabolites of tunicate-derived fungi, we isolated two novel sesquiterpenoid compounds, designated as JBIR-27 (1) and -28 (2), from the culture broth of Penicillium sp. SS080624SCf1 (Figure 1). In addition, we also isolated known derivatives, sporogen-AO1 8 and phomenone 9 (Figure 1). This paper describes the fermentation, isolation and brief biological activity of 1, 2, sporogen-AO1 and phomenone, in addition to the taxonomy of the producing microorganism. The structure elucidation of 1 and 2 is also reported.