Microbial urease (amidohydrolase, EC 3.5.1.5) is a metalloenzyme of significant biological and ecological importance, widely distributed among various microorganisms including bacteria, fungi, and archaea. This enzyme catalyzes the hydrolysis of urea (CH4N2O) into ammonia (NH3) and carbon dioxide (CO2), at a rate approximately 1014 times the rate of the noncatalyzed reaction. Urease is an enzyme that contains nickel and has a high molecular weight and many subunits. The cellular localization of urea necessitates its entry into the cell for consumption, and in certain organisms, energy-dependent urea absorption mechanisms have been recognized. The kinetic and thermodynamic mechanisms governing urease activity, emphasizing the enzyme’s active site dynamics, substrate binding, and catalytic efficiency. We discuss recent advances in understanding the molecular basis of urease function, including its thermodynamic stability and inhibition strategies. The presence and activity of urease are pivotal in both environmental and clinical settings, influencing soil fertility, plant growth, alcoholic beverage production, biocalcification, and human health. In agriculture, microbial urease contributes to soil alkalization and nutrient availability, making it a key factor in sustainable farming practices. This review delves into the thermodynamics and catalytic mechanism of microbial urease, highlighting the enzyme's active site, metal ion cofactors, and substrate specificity. We explore the regulation of urease expression in various microorganisms and discuss the environmental conditions that influence urease activity. Additionally, this paper provides a comprehensive understanding of microbial urease, emphasizing its dual role as both a beneficial and pathogenic agent in different ecosystems. The knowledge presented here aims to inform the development of strategies for managing urease activity in both environmental and clinical settings, with potential implications for improving human health and agricultural productivity.