As traditional cancer therapy is toxic to both normal and cancer cells, there is a need for newer approaches to specifically target cancer cells. ZnO nanoparticles can be promising due their biocompatible nature. However, ZnO nanoparticles have also shown cytotoxicity against mammalian cells in some cases, because of which there is a need for newer synthesis approaches for biocompatible ZnO nanoparticles to be used as carrier molecules in drug delivery applications. Here, we report the biosynthesis of ZnO nanoparticles using different plant parts (leaf, seed, and seed coat) of Bixa orellana followed by different characterizations. The UV−visible spectra of ZnO showed absorption maxima at 341 and 353 nm, 378 and 373 nm, and 327 and 337 nm, respectively, before and after calcination corresponding to the band gap energy of 3.636 and 3.513 eV, 3.280 and 3.324 eV, and 3.792 and 3.679 eV for L-ZnO, S-ZnO, and Sc-ZnO, respectively. Xray diffraction analysis confirmed the formation of hexagonal wurtzite structures. Attenuated total reflectance infrared spectra revealed the presence of stretching vibrations of C−C, CC, CO, and NH 3 + groups along with C−H deformation involving biomolecules from extracts responsible for reduction and stabilization of nanoparticles. Field emission scanning electron microscopy and transmission electron microscopy images showed spherical and almond-like morphologies of L-ZnO and Sc-ZnO with spherical morphologies, whereas S-ZnO showed almond-like morphologies. The presence of antibacterial activity was observed in L-ZnO against Staphylococcus aureus and Bacillus subtilis, in S-ZnO nanoparticles only against Escherichia coli, and in Sc-ZnO only against Staphylococcus aureus. Uncalcinated ZnO nanoparticles showed weak antibacterial activities, whereas calcinated ZnO nanoparticles showed a non-antibacterial nature. The antifungal activity against different fungi (Penicillium sp., Aspergillus flavus, Fusarium oxysporum, and Rhizoctonia solani) and cytotoxicity against HCT-116 cancer cells were not observed before and after calcination in all three ZnO nanoparticles. The antimicrobial nature and biocompatibility of ZnO nanoparticles were influenced by different parameters of the nanoparticles along with microorganisms and the human cells. Non-antimicrobial properties of ZnO nanoparticles can be treated as a pre-requisite for its biocompatibility due to its inert nature. Thus, biosynthesized ZnO nanoparticles showed a nontoxic nature, which can be exploited as promising alternatives in biomedical applications.