This study reports the synthesis of niobium diselenide (NbSe) nanoparticles at room temperature (RT), 70 °C, and 100 °C a sonochemical method, followed by an assessment of their structural, morphological, antimicrobial, cytotoxic, and wound healing properties. X-ray diffraction, scanning electron microscopy, and transmission electron microscopy analyses revealed changes in the crystallinity, size, and morphology of the nanoparticles as a function of synthesis temperature. The sample synthesized at 70 °C exhibited the highest cytotoxicity against HaCaT cells, while those synthesized at RT and 100 °C showed moderate and low cytotoxicity, respectively. Antimicrobial properties varied: the RT nanoparticles showed the highest inhibition against , while the 100 °C nanoparticles demonstrated some inhibition against . Wound healing assays revealed that the RT synthesized nanoparticles promoted the highest wound closure (94.78%) at a concentration of 0.5 μg mL. These results highlight the influence of synthesis temperature on the biological properties of NbSe nanoparticles, suggesting their potential for therapeutic applications in regenerative medicine, with the RT sample showing the most promising outcomes.