Two new polyketides, penifellutins A (1) and B (2), possessing a 22 carbon linear skeleton, were isolated from a co-culture of the deep-sea-derived fungi Penicillium crustosumPRB-2 and Penicillium fellutanumHDN14-323. Meanwhile, two esterification products of 1, penifellutins C (3) and D (4), were obtained because compound 1could be esterified spontaneously when stored in methanol. Their configurations were difficult to determine because of chiral central crowdedness, structural flexibility and instability. As such, we solved this issue by comprehensively using Mo2(OAc)4-based CD experiments, density functional theory calculation of 13C NMR, DP4 + probability analysis and many chemical reactions, including making acetonide derivative, Mosher’s method, PGME method, etc. Compounds 1and 2show obvious inhibitory activity on the liver hyperplasia of zebrafish larvae at a concentration of 10 μmol/L, while 3and 4show no activity, indicating that two carboxyls in the structure are important active sites.