Riboflavin, a water-soluble vitamin, plays a crucial role as a cofactor in various biochemical processes. Recently, it has gained attention for its potential use as a photosensitizer in antimicrobial and anticancer therapies. Riboflavin, upon exposure to ultraviolet A (UVA) light, generates reactive oxygen species (ROS), which are capable of damaging cellular structures, leading to microbial inactivation and cancer cell destruction. This book chapter explores the properties of riboflavin as a photosensitizer, highlighting its ability to produce singlet oxygen and other ROS that contribute to its therapeutic efficacy. Furthermore, the chapter also discusses the application of riboflavin in photodynamic therapy (PDT) for microbial infections and tumor cell destruction, comparing its effectiveness to other photosensitizers. Despite its advantages, such as low toxicity and endogenous presence, riboflavin’s employment in photodynamic therapy faces challenges like optimizing its concentration, light exposure, and long-term safety. This chapter also reviews the future potential of riboflavin as a photosensitizer.