Glucosinolates (GSLs) are sulfur-containing compounds predominantly found in cruciferous vegetables such as broccoli, kale, and Brussels sprouts, and are recognized for their health-promoting properties. Upon consumption, GSLs undergo hydrolysis by the enzyme myrosinase, resulting in bioactive compounds like isothiocyanates and specific indole glucosinolate degradation products, such as indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM), which contribute to a range of health benefits, including anti-cancer, anti-inflammatory, and cardioprotective effects. This review explores the structure, metabolism, and bioavailability of GSLs. Recent evidence supports the protective role of GSLs in chronic diseases, with mechanisms including the modulation of oxidative stress, inflammation, and detoxification pathways. Furthermore, the innovative strategies to enhance GSL bioactivity, such as biofortification, genetic introgression, and optimized food processing methods, have been examined. These approaches seek to increase GSL content in edible plants, thereby maximizing their health benefits. This comprehensive review provides insights into dietary recommendations, the impact of food preparation, and recent advances in GSL bioavailability enhancement, highlighting the significant potential of these bioactive compounds in promoting human health and preventing chronic diseases.