Photodynamic therapy (PDT) has recently become significant as a clinical modality for cancer therapy and multidrug-resistant (MDR) infections, replacing conventional chemotherapy and radiation therapy protocols. PDT involves the excitation of certain nontoxic molecules called photosensitizers (PS), applying a specific wavelength of light to generate reactive oxygen species (ROS) to treat cancer cells and other pathogens. Rhodamine 6G (R6G) is a well-known laser dye with poor aqueous solubility, and lower sensitivity poses an issue in using PS for PDT. Nanocarrier systems are needed to deliver R6G to cancer targets since PDT requires a higher accumulation of PS. It was found that R6G-conjugated gold nanoparticles (AuNP) have a higher ROS quantum yield of 0.92 compared to 0.3 in an aqueous R6G solution, increasing their potency as PS. Cytotoxicity assessment on A549 cells and antibacterial assay on MDR Pseudomonas aeruginosa collected from a sewage treatment plant are the evidence to support efficient PDT. In addition to their enhanced quantum yields, the decorated particles are effective in generating fluorescent signals that can be used for cellular imaging and real-time optical imaging, and the presence of AuNP is a valuable addition to CT imaging. Furthermore, the fabricated particle exhibits anti-Stokes properties, which makes it suitable for use as a background-free biological imaging agent. As a result, R6G-conjugated AuNP is an effective theranostic agent that prevents the progression of cancer and MDR bacteria, along with contrasting abilities in medical imaging with minimal toxicity observed in in vitro and in vivo assays using zebrafish embryos.