In the literature, only two papers describe the synthesis of 3-mercaptocoumarins (3-MCMs) without exploring their bioactivity. So, the novelty of this work is driven by the fact that it employs an original synthetic route to produce new 3-MCMs, assesses their biomedical activities, and compares them to those of similar 3-mercapto-free coumarins. By treating it with concentrated H2SO4, 1‑chloro-2-hydroxypropane-1,2,3-tricarboxylic acid was transformed into 2‑chloro-3-oxopentanedioic acid. The latter acid was sonicated separately with two derivatives of 2-naphthol to produce the precursors P-MCM1 and P-MCM8. These were in turn nucleophilically attacked by KSH at electrophilic carbon attached to chloride. The resulting two compounds, MCM1 and MCM8, were esterified individually with various parafunctionalized phenols, affording two series of 3-MCMs, which are coded MCM2-MCM7 and MCM9-MCM14. The structural frameworks of the synthesized MCM1-MCM14 were confirmed by examining their FTIR, 1HNMR , 13CNMR , and HRMS electromagnetic spectra. The biomedicine-related activities of the synthesized 3-MCMs were evaluated and compared with those acquired from their corresponding 3-mercapto-free coumarins (CM1-CM14). These activities include antitumor, cytotoxicity, antioxidant, antidiabetic, anti-aerobic bacteria, anti-anaerobic bacteria, antifungal, and microbiome biosafety. The MTT-probing methodology was used to assess the antitumor and cytotoxicity against six tumor cell lines and one healthy cell line, respectively. The hydroxyl‑ and DPPH radical-scavenging capacity of the synthesized 3-MCMs was assessed for their antioxidant activity. To quantify the antidiabetic activity, the inhibitory properties against two carbohydrate-metabolizing enzymes were followed. On the other hand, the broth microdilution methodology was employed to investigate the antimicrobial features against six pathogenic aerobes, four pathogenic anaerobes, and two pathogenic fungi. The microbiome biosafety was also inspected using the same methodology for one non-pathogenic aerobic bacterial strain. The results acquired from the above-mentioned activities regarding the synthesized 3-MCMs were analyzed and compared with those related to CMs. Collectively, the findings indicated that the synthesized 3-MCMs can demonstrate better biomedicine-related activities than those of CMs, with the exception of the anti-anaerobic one.