In this study, we report on the biosynthesis of gold nanoparticles (AuNPs) by using Callistemon citrinus (C. citrinus) seed extract as both reducing and capping agent as well as its characterization. Likewise, the in vitro antiparasitic activities of both biosynthesized gold nanoparticles and crude seed extract of the plant were evaluated. The formation of biosynthesized AuNPs was confirmed by a color change immediately when the seed extract was added to the gold chloride (III) solution. Characterization of the AuNPs was done using analytical tools like ultraviolet-visible spectroscopy, X-ray diffraction, scanning electron microscopy (SEM), energy dispersive X-ray (EDX), transmission electron microscopy (TEM) and Fourier transformed infra-red (FTIR). FTIR showed an absorption peak at 230 nm consistent with the absorption band for gold nanoparticles, the morphology and composition of AuNPs was ascertained by SEM and EDX micrographs; uneven spherical-shaped nanoparticles was established by SEM analysis, and an average particle size of about 37 nm was confirmed by the TEM analysis. The crude seed extracts exhibited antitrypanosoma activities with an IC50 of 11.06 µg/mL. Both the crude seed extract and AuNPs were inactive against plasmodial parasite, while the antibacterial assay showed that AuNPs is potent against gram positive and gram negative bacterial strains.