This chapter provides a comprehensive overview of recent advances in the preparation and biological applications of 1,2,4-thiadiazole, a versatile heterocyclic compound with diverse pharmacological properties. This chapter begins with the exploration of various synthetic strategies, ranging from traditional methods to sustainable and green approaches, highlighting their impact on synthetic efficiency and environmental considerations. The focus is then shifted to the biological applications of 1,2,4-thiadiazole derivatives, which show pharmacological activities, such as antioxidant, antimicrobial, anti-inflammatory, and anticancer properties. Structure–activity relationship studies are discussed which offer valuable insights into the molecular features influencing the bioactivity of these compounds. The relevance of 1,2,4-thiadiazoles in addressing contemporary health challenges is emphasized, and the potential of this scaffold for drug design and development is underscored. The documented pharmacological effects of 1,2,4-thiadiazole derivatives highlight their promising role in the development of therapeutic agents with enhanced efficacy and selectivity. The optimization of synthetic routes, scalability improvements, and exploration of innovative drug delivery systems are identified as crucial areas for future investigation. Ultimately, this chapter positions 1,2,4-thiadiazole as a pivotal candidate in the medicinal field, poised to make significant contributions to the pharmaceutical landscape.