The 4-anilinoquinazoline sulfonamide derivatives in medicinal chemistry are well-known antitumor backbones via different mechanisms. Herein, we reported the synthesis of five new 2,4-disubstituted-6-iodoquinazoline derivatives coded compounds 3a-e. The respective compounds were examined for their antiproliferative, antimicrobial, and carbonic anhydrase XII (CAXII) inhibitory activity against four cancerous cell lines. Compound 3c was proven to be the most effective as an anticancer. It stopped the growth of the four used tumor cell lines at concentrations ranging from 4.0 to 8.0 µM as compared to the reference doxorubicin (2.3-3.25 µM). Compound 3b, was able to stop the proliferation of the tumor cell lines with IC between 6.0 and 9.0 µM. The five compounds exhibited both broad-spectrum and antifungal action; however, their antibacterial activities against Gram-positive bacteria were superior to those of Gram-negative. Compound 3c showed the strongest antibacterial and antifungal activities, followed by compound 3b. In conclusion, the 4-substituted-anilino derivatives equipped with sulfonamide at position 4 of the quinazoline nucleus are suitable antitumor lead compounds. Moreover, the 4-methoxyphenyl substituent at position-2 has a better impact on activity than unsubstituted or even other substituents. The lab results are aligned with molecular docking analysis of the respective compounds against the potential targets, including CAXII, human thymidylate synthase (hTS), and human thymidine kinase (hTK), for the anticancer activities and DHFR of E. coli and S. aureus for the antibacterial properties. Compounds 3c and 3b are highly recommended for preclinical and clinical evaluation as anticancer and/or antibacterial agents for potential use in humans.