Nowadays, the growth of drug-resistant microbial strains (MDRs) is a serious public health threat worldwide. Moreover, tens of millions of people are annually diagnosed with cancer worldwide, and more than half of patients ultimately die. In the present study, a new series of 2-(4-substituted-thiazol-2-ylamino)acetamides and N-(4-substituted-thiazol-2-yl)acetamides incorporating sulfonamide moieties were designed, synthesized, well-characterized and successfully evaluated for their antimicrobial activity against multidrug resistant strains and screened for cytotoxic activity against normal lung fibroblast (WI-38), human lung carcinoma (A549), and human breast carcinoma (MDA-MB-231) cell lines. Fluorescence-activated cell sorting (FACS) analysis and molecular modeling study were performed to identify the mode of action of the novel synthesized compounds and their binding interactions with the active sites of dihydrofolate reductase enzyme (DHFR).