Environmentally benign-synthesized gold nanoparticles (Au-NPs) have received substantial attention owing to their biomedical applications, particularly in cancer therapy. In the current study, Backhousia citriodora (B. citriodora) leaf extract was applied as a reducing agent for one-pot synthesis of controlled size Au-NPs. The effect of various parameters such as reaction time, pH, and B. citriodora leaf boiling time on the synthesis of Au-NPs was studied. The characterization of the Au-NPs synthesized at 15.0-min incubation time showed colour change because of the surface plasma resonance band around 530.0 nm. TEM photographs showed spherical morphologies with an average size of 8.40 ± 0.084 nm and zeta potential value was − 29.74 mV, indicating stability of the nanoparticles. The biomedical properties of Au-NPs and B. citriodora leaf extract showed strong DPPH radical scavenging. The in vitro anticancer activity determined using MTT assay exhibited that Au-NPs showed a significant dose-dependent reduction in the viability of the MCF-7 breast cancer cell line and the HepG2 liver cancer cell line with IC50 values of 116.65 and 108.21 µg, respectively.