In this study, we report the synthesis, structural and morphological characteristics of zinc oxide (ZnO) nanoparticles using solution combustion synthesis method where lemon juice was used as the fuel. In vitro anti-tubercular activity of the synthesized ZnO nanoparticles and their biocompatibility studies, both in vitro and in vivo were carried out. The synthesized nanoparticles showed inhibition of Mycobacterium tuberculosis H37Ra strain at concentrations as low as 12.5 μg/mL. In vitro cytotoxicity study performed with normal mammalian cells (L929, 3T3-L1) showed that ZnO nanoparticles are non-toxic with a Selectivity Index (SI) > 10. Cytotoxicity performed on two human cancer cell lines DU-145 and Calu-6 indicated the anti-cancer activity of ZnO nanoparticles at varied concentrations. Results of blood hemolysis indicated the biocompatibility of ZnO nanoparticles. Furthermore, in vivo toxicity studies of ZnO nanoparticles conducted on Swiss albino mice (for 14 days as per the OECD 423 guidelines) showed no evident toxicity.