It is very important to use the green synthesis approach that uses living things and plants. Using the biogenic reduction technique, silver nanoparticles were synthesized from the R. aculeatus plant for this research. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, X-ray diffraction (XRD), and UV-vis spectroscopy was used to characterize the produced AgNPs (FT-IR). After the material characterization of NPs, their antibacterial properties were investigated against 15 different bacterial strains. Disk diffusion and microdilution techniques were used in the examination. In the disk diffusion study, the extract and AgNP gave the best results. In plant extract studies, Bacillus subtilis and Enterobacter aerogenes ATCC 13048 had the highest antibiofilm activity, whereas Staphylococcus aureus ATCC 25923 had the lowest. The Salmonella infantis bacterial strain showed the maximum action on AgNP, whereas Escherichia coli CFAI ATCC 25922 had the lowest. At the concentrations established in cytotoxic activity experiments, the plant-AgNP complex had the lowest concentration value on MCF-7 and HUVEC cell lines of 10-3 g/mL. There was no cytotoxic effect of the extract on the MCF-7 cell line. It had a cytotoxic activity of 10-3 g/ml at the lowest concentration in the HUVEC cell line. An important research result was obtained for the investigation of the antibacterial, antibiofilm, and anticancer effects of R. aculeatus plant extract and biogenically derived AgNPs. The comparative study on both breast cancer cell lines and healthy cell lines in the area of cell culture offers a wide range of resources for science in addition to the comparative data of the study done based on both plant extract and AgNP.