A series of functionalized sesquiterpenoids derived from benzocyclooctene, including natural parvifoline (1), isoparvifoline (3), epoxyparvifoline (5), epoxyisoparvifoline (7), 8,12-oxyparfivoline (9), 8,14-oxyparvifoline (11), and the respective benzoyl derivatives 2, 4, 6, 8, 10, and 12, were prepared and tested for their inhibitory effect on the in vitro α,β-tubulin polymerization process. The structural analysis and characterization of the new compounds 5–7 and 9–12 were achieved by 1D and 2D NMR spectroscopy, mass spectrometry, and X-ray diffraction analysis of 6, 7, and 9. Preparation of 9 and 12 involved molecular rearrangements of the epoxide group with transannular 1,5-hydride shifts. At 10 μM compounds 1, 5, and 8 inhibited the polymerization of the α,β-tubulin heterodimer by 24%, 49%, and 90% as compared to colchicine. These compounds were subjected to docking analysis that supported their interactions in a colchicine binding site located in the α-tubulin subunit, in the pocket formed by Phe296, Pro298, Pro307, His309, Tyr312, Lys338, Thr340, Ile341, and Gln342. Competitive inhibition assays with colchicine were also performed for the three compounds, which supported their binding at the colchicine secondary site in α-tubulin. Also, evaluations of their cytotoxicity on MCF7 breast carcinoma, HeLa cervix carcinoma, and HCT 116 colon carcinoma cell lines were carried out and showed that 8 is active against the HeLa and HCT 116 cell lines with IC50 3.3 ± 0.2 and 5.0 ± 0.5 μM, respectively.