<p>Cu₂SnS₃ (CTS) is potent substitute for conventional compounds due to adjustable bandgap, conductivity of p-type, adaptable morphology, easy to synthesize, and superior thermoelectric properties. In this work, the CTS nano-spheres are synthesized by hydrothermal technique. The synthesized CTS nano-spheres are employed for the antibacterial, antioxidant, and cytotoxic application. Structural analysis by X-ray diffraction confirms cubic unit cell structure of CTS nano-spheres. The energy-dispersive X-ray analysis showed CTS nano-spheres to be copper-rich and tin-deficient. Scanning electron microscopy images revealed nano-spheres with needle-like surface features. The CTS nano-spheres possess direct bandgap of 1.58&#xa0;eV, confirmed by diffuse reflectance spectroscopy. The antibacterial activity shows 100% activity index with higher zone of inhibition in <i>Listeria monocytogenes</i> and <i>Staphylococcus aureus.</i> The antioxidant activity of CTS nano-spheres determined using the DPPH assay showed IC₅₀ value of 61.60&#xa0;µg/ml stating moderate antioxidant efficiency. The in vitro cytotoxic analysis is carried out by employing A549 lung cancer cell lines. The in vivo and vitro cytotoxic analysis provided the potent cytotoxicity of CTS nano-spheres, as reflected in LC₅₀ value of 40.40&#xa0;µg/ml and IC₅₀ value of 57.75 ± 2.34&#xa0;μg/ml. The mechanistic evolution of CTS nano-spheres for their antibacterial activity is proposed in this work. The leaching behaviour of CTS nano-spheres revealed higher leaching of Sn⁴⁺ ions than Cu⁺ ions, contributing to their strong antibacterial activity. The zeta potential of CTS nano-spheres is found to be − 30.70&#xa0;mV, which showed less agglomeration of CTS nano-spheres, depicting an efficient antibacterial activity. The obtained results are rigorously analysed and supported with relevant data.</p>