Shape, size, and homogeneity affect the biological activity of gold nanoparticles (AuNPs) in nanomedicine and catalytic applications. Here we biosynthesized monodispersed isotropic and polydispersed anisotropic spherical AuNPs from leaf and seed extract broths of the medicinal plant Peganum harmala L. (Ph. L). Synthesized AuNPs were characterized by ultraviolet-visible spectroscopy, Fourier-transform infrared spectroscopy (FT-IRS), field-emission scanning electron microscopy (FESEM), X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDX), and transmission electron microscopy (TEM). The antimicrobial activity of AuNPs against Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) human pathogens was also assessed. Leaf- and seed-derived AuNPs had characteristic localized surface plasmon resonances of 530 and 578 nm, respectively. TEM, FE-SEM, EDX, and XRD revealed the formation of elemental face-centered cubic spherical monodispersed isotropic AuNPs of average size 43.44 nm and polydispersed anisotropic AuNPs of average size 52.04 nm from leaf and seed extract broths, respectively. FT-IR revealed polyphenols and alcohols as responsible for AuNP capping, reduction, and protection. Anisotropic AuNPs showed no antibacterial activity, whereas isotropic AuNPs showed good inhibition of both E. coli and S. aureus. This represents a simple and ecofriendly protocol for the green synthesis of monodispersed isotropic spherical AuNPs, which may have value in a variety of applications.