Natural sources, particularly plant sources, have been the main source of medicinal compounds for thousands of years. Azadirachta indica, commonly known as neem in India, is renowned for its extensive array of bioactive compounds, particularly alkaloids, that remain the focus of substantial research for their potent biological activities. It is a tree indigenous to the Indian subcontinent, widely recognized for its therapeutic and medicinal properties. Neem seeds contain several bioactive alkaloids, with azadirachtin being the most prominent. Other notable alkaloids include nimbin, nimbidin, and salannin. These compounds are characterized by complex chemical structures that contribute to their diverse biological activities. Neem seed alkaloids exhibit a broad spectrum of pharmacological properties that render their anti-microbial, anti-inflammatory, anti-cancer, and immunomodulatory effects. These properties position neem as a valuable resource in developing novel therapeutic agents and biotechnological applications. This chapter delves into the diverse alkaloids present in neem seeds, the multifaceted biological potential present within them, and their biotechnological applications, preferably in agriculture and pharmaceutical industries. This chapter would be significant in indicating the future research directions to validate the therapeutic potential of these alkaloids.