Singlet oxygen (1 O2 ) is an excited form of molecular oxygen that acts as an active agent in antimicrobial Photodynamic Therapy (PDT), a promising technique for the treatment of several types of infections. In this review, we present a summary of the physical-chemical and biological aspects of 1 O2 and the lethal photosensitization mechanisms mediated by it in PDT. The chemical structure of H2 O will be approached through molecular orbital theory. PDT techniques that enhance the formation of this radical using different photosensitizing dyes will also be introduced as well as biological targets of 1 O2 in microbial cells and the obstacles encountered in such structures. This knowledge is crucial to design new research methodologies that increase the chain of reaction of 1 O2 in PDT, consequently, increase the effectiveness of the therapy