Chemical investigations on the culture of Penicillium oxalicum 2021CDF-3, a marine red alga-sourced endophytic fungus, revealed a new steroid, namely peniciloxatone A (1). With the aid of high-resolution electrospray ionization mass spectrometry (HRESIMS) and nuclear magnetic resonance (NMR) analyses, the structure of peniciloxatone A (1) was determined to be a polyoxygenated ergostane steroid. Cytotoxic activities of 1 were tested against A549, FADU, and HepG2 cells, and 1 was active against the FADU and HepG2 cells, with IC50 values of 9.5 ± 0.1 and 18.1 ± 0.3 µM, respectively.