Antimicrobial peptides (AMPs) and their conjugation with nanoparticles hold promise for targeting Methicillin-resistant Staphylococcus aureus (MRSA). In this study, Jellein-I peptide, with a cysteine at the C-terminus, was conjugated to gold nanoparticles (GNPs) to enhance its antibacterial, antibiofilm, and anticancer activities. The GNPs were synthesized and then conjugated to Jellein-I, forming Jellein-I-Cys-GNPs. The nanoparticles were characterized using UV-visible spectroscopy, transmission electron microscopy (TEM), dynamic light scattering (DLS), and Fourier transform infrared (FTIR) spectroscopy. UV-visible spectroscopy, DLS, and TEM confirmed the formation of GNPs with an average size of 5.5 nm. The UV–visible spectroscopy and FTIR results indicated the presence of peptide functional groups on the surface of the GNPs. To assess antibacterial activity, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays were performed against MRSA strains. The conjugated Jellein-I exhibited lower MIC and MBC values (94 μM) compared to the free peptide (512 μM) against both clinical and standard MRSA strains. Additionally, GNP-conjugated Jellein-I demonstrated antibiofilm activity, inhibiting and removing biofilms while reducing metabolic activity by more than 82%, 44%, and 72%, respectively. Cell viability studies on rat dermal fibroblast (RDF) cells showed over 80% viability across all treatment concentrations. Furthermore, a clonogenic assay on the metastatic melanoma cell line (A375) demonstrated that GNP-conjugated Jellein-I had significantly better anticancer effects than the free peptide, even at a concentration as low as 9 μM. Our findings suggest that the conjugation of Jellein-I-Cys to GNPs could be a promising formulation for enhancing the antibacterial, antibiofilm, and anticancer activities of peptides. Further in vivo studies may establish this new nanoformulation as a potential therapeutic candidate for MRSA infections and cancer treatment.