Azetidin-2-ones (β-lactams) have been a mainstay as therapeutics for the treatment of a wide range of bacterial infections. The interest in β-lactams stems from their highly unusual structure-activity profiles towards a genera of pathogenic bacteria. Tremendous efforts have been made in the literature towards the development of novel β-lactam compounds for a study of their biological evaluation. The present review begins with a brief introduction of β-lactam antibiotics. This is preceded by the progress made towards the biological evaluation of a variety of racemic as well as optically active cis- or trans-3-acetoxyazetidin-2-ones. Their application in medicinal field as antifungal, antibacterial, antitumor, anticancer, IMR32 cytotoxic agents and carbonic anhydrase inhibitors is discussed.