Silver nanoparticles (AgNPs) have several uses. Many scientists are working on producing AgNPs from plant extracts for use as biomedicines against drug-resistant bacteria and malignant cell lines. In the current study, plant-based AgNPs were synthesized using Raphanus sativus L. (RS) leaf aqua extract. Different concentrations of AgNO3 were used to optimize the synthesis process of RS-AgNPs from the aqueous leaf extract. Energy-dispersive X-ray analysis (EDX), transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscope (AFM), and UV-vis spectroscopy were used to analyze the generated materials. Furthermore, to evaluate the biological properties of the obtained materials, Bacillus subtilis (B. subtilis), Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Candida albicans (C. albicans) pathogen strains were used for the minimum inhibitory concentration (MIC) assays. Subsequently, healthy cell lines (human dermal fibroblast (HDF)) and cancerous cell lines (glioma/U118, Ovarian/Skov-3, and colorectal adenocarcinoma/CaCo-2) were engaged to determine the cytotoxic effects of the synthesized NPs. The cytotoxic and anti-pathogenic potential of AgNPs synthesized by the proposed green approach was investigated. The results were encouraging compared to the standards and other controls. Plant-based AgNPs were found to be potential therapeutic agents against the human colon cancer cell (CaCo-2) and showed strong inhibitory activity on Candida albicans and Staphylococcus aureus growth. The RS-AgNPs generated have highly effective antimicrobial properties against pathogenic bacteria. Our findings also show that green RS-AgNPs are more cytotoxic against cancerous cell lines than normal cell lines. Synthesized nanoparticles with desirable morphology and ease of preparation are thought to be promising materials for antimicrobial, cytotoxic, and catalytic applications.