Introduction/Objective: Biguanide derivatives are small molecules with promising antitumor activity. However, the effect of different carbon rings at the end of one guanide group of these compounds on anti-proliferation activity is unknown. Therefore, we synthesized novel fluorine- containing biguanide compounds with various carbon rings, evaluated their anticancer activities, and explored their anti-proliferative mechanisms. Methods: Guanidine derivatives containing trifluoromethoxy or 3,4-difluorophenyl with nine different carbon rings were synthesized using established chemical methods. The phenyl side chain was fixed to trifluoromethoxy or 3,4-difluorophenyl with changes in the number of cyclic aminocyclic carbons. The effects of these derivatives were evaluated using MTT and clonogenic assays, while the underlying mechanisms were investigated by analyzing protein expression levels via western blotting. Results: This study analyzed the effects of new biguanide derivatives on cell growth in three different cell lines: HepG2, Ovcar3, and T24. The results showed that T24 cells were the most sensitive cell line to these biguanides. All biguanide derivatives significantly inhibited the growth of T24 cells, while compound 4b exhibited the strongest inhibition in all three cell lines by MTT assay. The inhibitory effects of 4b were further confirmed using colony formation experiments. Western blotting results indicated that the representative biguanide derivative, 4b, inhibited the EGFR signaling pathway, thereby inhibiting tumor growth. Conclusion: 1a-5a and 1b-4b, the cyclooctyl-containing 3,4-difluorophenyl biguanide analogs, have demonstrated significant potential in developing novel anticancer drugs. The 3,4- difluorophenyl biguanide containing cyclooctyl showed the best antitumor activity among the nine derivatives. This finding offers a novel perspective in developing anticancer drugs and a further improvement in biguanide activity in the future.