A new series of 2-amino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carbonitrile (ATCTC) schiff bases were synthesized and evaluated against Mycobacterium tuberculosis H37Rv strain and clinically isolated XDR-TB strains. Our findings exhibited that compounds 2a, 2d, 2j, and 2 m showed higher potency against Mtb.H37Rv and extensively drug-resistant TB, with MIC values ranging from (0.04 - 0.14 µgm/mL) and (0.017–0.29 µgm/mL), respectively, compared to 297F with MIC (0.23 µgm/mL). Furthermore, all synthesized compounds were tested for in vitro anticancer activity against the HepG2, NCI-H460, and MCF cancer cell lines. Our findings demonstrated that compound 2 m had the maximum activity against all three cell lines, whereas compounds 2a, 2d, and 2e had moderate activity against all of the investigated cell lines compared to the doxorubicin as reference. Additionally, docking experiments shown excellent results in inhibiting VEGFR-2, EGFR, ERα, CDK2, and DNA topoisomerases Type II. The results showed that compound 2 m was the most effective compound, with an inhibitory effect comparable to that of the reference medications Doxorubicin. The molecular docking investigation supported the biological finding. Compounds (2 m) had the highest affinity for VEGFR-2, EGFR, ERα, CDK2, and DNA topoisomerases II, with binding affinity of -10.30, -10.10, -8.90, -8.30, and -9.20, respectively, compared to binding affinity of Doxorubicin (-7.90, -10.50, -8.10, -8.20, and -9.00, respectively). Furthermore, our findings revealed that compounds 2a, 2d, and 2 m of M. tuberculosis FtsZ protein with binding energies (-8.90, -9.50, and -8.90) had a higher affinity than (297F -8.10). Also, Physicochemical properties, drug-likeness, and ADME (absorption, distribution, metabolism, excretion, and toxicity) parameters of the selected compounds were also computed. These findings imply that these compounds have the potential to be developed as anticancer agents and enzyme inhibitors for the treatment of M. tuberculosis and associated illnesses.