Silver nanoparticle investigations and product development assist in targeted drug delivery. The aqueous extract (PAFA) of Pimpinella anisum L. fruits was prepared, and its constituents were identified using UPLC-ESI-MS/MS. Silver nanoparticles were synthesized using PAFA via microwave irradiation. The prepared AgNPs were characterized using UV‒visible spectroscopy, TEM, FTIR, EDX, and XRD. The PAFA and AgNPs were investigated for their cytotoxicity against 5 different cell lines: Colo-205, HepG-2, H-460, MCF-7, and OEC. In addition, the antibacterial effects of AgNPs and their prepared topical creams were assessed. Thirteen compounds were tentatively identified, in addition to the successful green synthesis and characterization of the dose-dependent and selective AgNPs possessing effective cytotoxicity against Colo-205. AgNPs and their topical creams showed potent antibacterial effects on a broad spectrum of organisms. Molecular docking analysis of the identified phytochemicals revealed that they are involved in a variety of cancers, including lung, breast, head, neck, colorectal, and pancreatic cancer. The best binding pose of the promising kaempferol-3-O-methyl ether inside the active site of EGFR is shown by 2D and 3D representations of the amino acid interactions inside the binding pocket. These results confirm that the prepared AgNPs of PAFA are promising for application as efficient cytotoxic agents against Colo-205 and as topical antimicrobial agents.