Biosynthesis of nanoparticles from plant extracts is of great interest because it is inexpensive, environmentally friendly, and suitable for large-scale production compared to other synthesis methods. In this study, it was aimed to perform the synthesis and characterization of Ag nanoparticles (Ag NPs) by using Catharantus roseus (C. roseus) extract as a reducing and stabilizing agent and to evaluate their toxic effects against a healthy human cell line. The synthesis of Ag NPs were successfully carried out using the extract of C. roseus. By using FTIR, XRD, UV-Vis, and TEM analysis, the synthesized Ag NPs were characterized. According to XRD and TEM analysis results, it was observed that the average diameter of the synthesized Ag NPs was 16 ± 6 nm and its shape was a face-centered cubic (fcc). In addition, the toxic effects of biologically synthesized Ag NPs against a human healthy cell line (L929) were investigated in the study and the half-maximum inhibitory concentration (IC50) was found to be 2.909 µL/mL.