Three new γ-pyrones named marinactinones A–C (1–3) were isolated from marine-derived actinomycete Marinactinospora thermotolerans SCSIO 00606. These structures were elucidated by extensive spectroscopic methods. All three new compounds were evaluated for cytotoxic effects on six cancer cell lines and inhibitory activities of DNA topoisomerase II. Compounds 1–3 exhibited moderate cytotoxicities against SW1990, HepG2 and SMCC-7721 cell lines, and compound 2 showed weak DNA topoisomerase II inhibition activity. This is the first report on the chemical constituents and their biological activities from Marinactinospora, a novel genus of marine actinomycetes.