A series of novel γ,γ-difluorinated Goniothalamin analogues 4a–4i and 6a–6i were synthesized. The key steps included the construction of C-5 stereocenter adjacent to gem-difluoromethylene group by way of lipase AK catalyzed kinetic resolution, the introduction of aryl group via Stille coupling, and lactonization by 1,5-oxidative cyclization. These γ,γ-difluorinated Goniothalamin analogues 4a–4i and their enantiomers 6a–6i, together with several corresponding γ-monofluorinated Goniothalamin analogues were biologically evaluated against four different cancer cell lines. Compound 7h showed a nearly equivalent potency as the parent (R)-Goniothalamin in the micromolar range. The different fluorine effects between fluoromethylene and gem-difluoromethylene on antitumor activity were discussed through the analysis of bioassay data.