Fungi have proven to be a very rewarding source of bioactive and structurally diverse natural products. In the course of our ongoing screening program aimed at the discovery of novel and selective antitumor agents potentially acting at molecular targets, we have discovered two new antitumor alkaloids, stephacidin A (1) and B (2). Compounds 1 and 2, produced by Aspergillus ochraceus WC76466, are structurally related to the cytotoxic marine natural product, avrainvillamide (3), isolated first from a marine fungal strain of Aspergillus sp. by Fenical and co-workers, and later from the fermentation broth of Aspergillus ochraceus by Sugie and co-workers under the name of CJ-17,665. Both compounds 1 and 2 demonstrated in vitro cytotoxic activity against various human tumor cell lines, but 2 exhibited more potent and selective antitumor activities, especially against testosterone-dependent prostate LNCaP cell line with an IC50 value of 0.06 µM. The structures of 1 and 2 (Figure 1) were established on the basis of NMR data and X-ray crystallography. In this paper, we report the isolation, characterization and preliminary in vitro antitumor screening of these two novel fungal alkaloids.