The cytotoxic activity of the pimarane diterpene annonalide (1) and nine of its semisynthetic derivatives (2–10) was investigated against the human tumor cell lines HL-60 (leukemia), PC-3 (prostate adenocarcinoma), HepG2 (hepatocellular carcinoma), SF-295 (glioblastoma) and HCT-116 (colon cancer). The preparation of 2–10 involved derivatization of the side chain of 1 at C-13. Among the derivatives, compounds 3-10 are being reported for the first time. Most of the tested compounds presented IC50 equal or lower than 4 µg/mL, being considered as promising antitumor drugs. The structures of all compounds were elucidated by spectroscopic analyses including 2D NMR and HRMS. Additionally, the interaction of annonalide (1) with ctDNA was evaluated through spectroscopic methods, and the formation of the supramolecular complex 1-ctDNA was proved. Through competition assays with fluorescent probes (Hoechst and ethidium bromide), it was found that 1 interacts preferentially via DNA intercalation.