This study investigated the secondary metabolites of the marine-derived Streptomyces aureus and their cytotoxic activities. The ethyl acetate extract of the fermentation products of S. aureus was separated by separation technologies such as silica gel column chromatography, Sephadex LH-20 colume, and semi-preparative HPLC. Their structures were identified by spectroscopic methods, combined with reference literature and quantum chemical calculations. Then, the cytotoxic activity of the separated compounds was tested. Finally, six compounds were isolated and identified from the ethyl acetate fraction of the fermentation broth of this bacterium, namely aurelactone A (1), (3S, 4S, 5S, 7S)-3-hydroxy methyl-7-methyl-1,6-dioxaspiro[4.5]-decan-4-ol (2), (3R, 4S, 5S, 7S)-3-hydroxymethyl-7-methyl-1,6-dioxaspiro[4.5]decan-4-ol (3), N-(tetrahydro-2-oxo-3-thienyl)-acetamide (4), 3-[(1-carboxy-vinyl)oxy] benzoic acid (5), and methyl-2-hydroxy-3-(4′-hydroxy)-phenylpropionate (6). Among them, compound 1 was a new spiroketal compound. The activity results showed that compound 2 had weak inhibitory activity against Hela cells, with an IC₅₀ of 70 ±1.5 μmol/L.