Light, a non-invasive tool integrated with cutting-edge nanotechnologies, has driven transformative advancements in imaging-based diagnosis and drug delivery for cancer and bacterial treatments. This review discusses recent progress in these areas, beginning with emerging imaging technologies. Unlike traditional photosensors activated by visible light, alternative energy sources such as near-infrared (NIR) light, X-rays, and ultrasound have been extensively investigated to activate various photosensors, achieving high sensitivity, wavelength versatility, and spatial resolution for deep-tissue imaging. Moreover, to address challenges like tissue autofluorescence in real-time fluorescence imaging, afterglow luminescent nanoparticles are being developed by integrating these alternative energy sources for real-time imaging and sensing in deep tissue for precise cancer diagnosis and treatment beyond superficial tissues. In addition to deep tissue imaging, light-responsive nanomedicines are revolutionizing anticancer and antimicrobial phototherapy by enabling spatially and temporally controlled drug release. These smart nanoparticles are engineered to release therapeutic cargo at target sites in response to microenvironmental cues specific to tumors or infections. In anticancer phototherapy, these nanoparticles facilitate controlled drug release via photoisomerization, photothermal, and photodynamic processes. To enhance circulation time and specific targeting, biomimetic nanoparticles, which mimic natural anti-tumor responses by our body, have attracted increasing attention. In antimicrobial phototherapy, research has been focused on the chemical modification of the photosensitizer to enable targeted drug delivery. An intriguing strategy has recently emerged involving the development of "pro-photosensitizers" that are specifically activated within bacterial cells upon light irradiation, offering a high margin of safety. These advancements leverage photochemical reactions and nanotechnology to enhance precision therapy and diagnosis in addressing critical health challenges.