Bioactivity of nanomaterials largely depends on its size, shape and crystalline nature. In this work, the smaller sized spherical shaped nano-zirconia (ZrO2 NPs) (of ~ 9 to 11 nm) was fabricated and studied its biological activity especially antioxidant and cytotoxicity against human colon carcinoma (HCT-116) and human lung carcinoma (A-549) cell lines. To have its real applications in biological aspects readily available Eucalyptus globulus (E. globulus) leaf extract was used as an effective capping and reducing agent for its synthesis. The prepared ZrO2 NPs was characterized by using different sophisticated instrumentations such as UV–visible spectrophotometer, XRD, FTIR, TEM, SAED, EDX, DLS and fluorescence spectroscopy. Cellular mitochondrial activity i.e. cell viability was measured by MTT assay and anti-oxidant activity was determined by DPPH assay. The smaller sized ZrO2 NPs showed strong antioxidant activity as well as cytotoxicity on human cancer cell lines. Comparative cytotoxic studies were conducted on human cancerous cell lines using different techniques. Results confirmed the efficient anti-cancer activities of the fabricated ZrO2 NPs towards the tested cell lines as well as efficient anti-oxidant activity. This is the first study in which E. globulus leaf extract was used to synthesize smaller spherical shaped ZrO2 NPs for improved bioactivity i.e. antioxidant and cytotoxicity. © 2017 Elsevier B.V.