Glutaminase, reported from various microorganisms such as bacteria, yeasts, and fungi, primarily catalyzes the hydrolysis of the γ-amido bond of L-glutamine to yield L-glutamic acid. Certain glutaminases also facilitate a γ-glutamyl transfer reaction, which is valuable for synthesizing bioactive compounds such as theanine, a flavor-enhancing amino acid naturally present in green tea. Owing to this unique functionality, microbial glutaminases are widely employed as flavor enhancers in the food industry. Despite these promising applications, a significant knowledge gap remains in understanding the structural characteristics of microbial glutaminases, particularly their primary and secondary structures. Additionally, this chapter explores the therapeutic applications of microbial glutaminases, examining their role in monotherapy and combination strategies for cancer treatment, as well as their potential in other diseases. This chapter attempts to provide insights into their physicochemical properties, thereby contributing to a deeper understanding of the structure–function relationship of microbial glutaminases and paving the way for their therapeutic and industrial applicability.