Plants have developed intricate chemical defense mechanisms to safeguard themselves against herbivores and pathogens. Among these, two secondary metabolites, glucosinolates (GSLs) and cyanogenic glycosides (CGs), are pivotal in bolstering plant defense. While CGs are present in diverse plant species, GSLs are predominantly found in the Brassicaceae family and are distinguished by their sulfur and nitrogen content. Both GSLs and CGs are nitrogen-containing compounds, and their synthesis involves complex biochemical pathways governed by enzymes and regulatory elements. Despite having different biosynthetic pathways to GSLs and CGs, the enzymes and regulatory factors involved in their synthesis share some similarities. Furthermore, both compounds are hydrolyzed in response to herbivory or tissue damage, releasing toxic breakdown products that detect herbivores and protect the plant from further harm. Although GSLs and CGs produce various toxic breakdown products, both are effective herbivore deterrents. The potential anticancer, anti-inflammatory, and antioxidant capabilities of GSLs and their breakdown products have attracted increasing attention in recent years. Similarly, there has been interest in using CGs as biopesticides. This chapter provides a detailed overview of the biosynthesis and mechanism of action of GSLs and CGs, focusing on the shared and distinct molecular mechanisms involved in their production and their roles in plant defense.