Exogenous 5-aminolevulinic acid (ALA) is taken up directly by bacteria, yeasts, fungi, and some parasites, which then induces the accumulation of protoporphyrin IX (PPIX). Subsequent light irradiation of PPIX leads to the inactivation of these organisms via photodamage to their cellular structures. ALA uptake and light irradiation of PPIX produced by host cells leads to the inactivation of other parasites, along with some viruses, via the induction of an immune response. ALA-mediated PPIX production by host cells and light irradiation result in the inactivation of other viruses via either the induction of a host cell response or direct photodynamic attack on viral particles. This ALA-mediated production of light-activated PPIX has been extensively used as a form of photodynamic therapy (PDT) and has shown varying levels of efficacy in treating conditions that are associated with microbial infection, ranging from acne and verrucae to leishmaniasis and onychomycosis. However, for the treatment of some of these conditions by ALA-based PDT, the role of an antimicrobial effect has been disputed and in general, the mechanisms by which the technique inactivates microbes are not well understood. In this study, we review current understanding of the antimicrobial mechanisms used by ALA-based PDT and its role in the treatment of microbial infections along with its potential medical and nonmedical applications.