A series of new fluorene-heterocyclic sulfonamide conjugates were designed and synthesized as potential anticancer agents. In the design of the conjugates, heterocyclic ring systems were utilized for a plausible amplification of bioactivity. The new fluorene-based conjugates were thoroughly characterized and eval- uated for antibacterial and anticancer activity against selected bacterial strains and cancer cell lines. The conjugate 8 g , with a 4,6-dimethyl-pyrimidinyl group, exhibited excellent cytotoxicity and selectivity in- dex of 5.6 μM (IC 50 ) and 10.14, respectively, against HCT-116 cancer cell line, which was comparable and superior to standard doxorubicin. Additional clonogenicity, cell migration, and apoptosis induction assays demonstrated that the conjugate 8 g effectively inhibits the colony forming and cell migratory ability of HCT-116 cancer cells with significant apoptosis induction. Moreover, in silico analysis was carried out to understand their binding affinity at the DHPS receptor and all the analyzed conjugates exhibited supe- rior or similar affinity towards the target protein compared to sulfamerazine drug, which was used as control. Additionally, the ADME pharmacokinetics predictions, along with drug likeliness properties, were also investigated.