Background: Nanoparticles (NPs) are small-sized particles with dimensions of 1 - 100 nm that have various medical and pharmaceutical technology applications. Objectives: In this study, we aimed to develop a quick, green, and eco-friendly procedure for the synthesis of gold NPs (AMGNPs) using the aqueous extract of Achillea millefolium (AM) and evaluate the effects of them on preantral follicle (PF) maturation in vitro compared to commercially provided GNPs (C-GNPs) and the AM extract alone. Methods: We assessed PF maturation through the morphological assessment of follicles, diameter changes, and estradiol and progesterone levels in PF. We also evaluated the morphometric indices in the control group and groups treated with the AM extract, green GNPs, and C-GNPs. Results: The results showed that the AM extract had positive effects on the PF development by increasing the production of estradiol. The evaluation of PF treated with C-GNPs one to four days after the treatment showed that the mean diameter of follicles was significantly reduced in the C-GNPs group compared to the control group. Moreover, the mean estradiol level increased, and the mean progesterone level decreased in all the experimental (10, 25, 50, and 100 µg/mL of C-GNPs, green GNPs, and the AM extract) groups compared to the control group (P-value < 0.05). The size and concentration of the NPs were 20 nm and 150 µg/mL, respectively. Conclusions: The findings suggest that green GNPs synthesized with the AM extract can minimize the hazardous effects of NPs and have beneficial effects on the development and growth of PF.