Abstract: Honey is a natural sweet substance made from a mixture of nectar collected by worker bees and their own secretions, which has good antibacterial activity and contains a variety of natural antibacterial active substances. In recent years, a large number of studies have been reported on the progress of antimicrobial active substances in honey, including methylglyoxal, hydrogen peroxide, polyphenols, antimicrobial peptides and Major royal jelly proteins. Numerous studies have shown that hydrogen peroxide is one of the most important antibacterial substances in honey. This paper highlights the important roles by each antimicrobial effective active substance for the anti-bacterial by honey. It also further summarizes the process of glucose oxidase catalyzing the production of hydrogen peroxide from glucose. The process of glucose oxidase catalyzing the production of hydrogen peroxide from glucose is summarized using hydrogen peroxide in honey as an example. Other pathways for the production and degradation of hydrogen peroxide and the physicochemical factors affecting the production of hydrogen peroxide are also reviewed. Rational regulation of hydrogen peroxide production in honey plays an important role in its antibacterial and even other biological activities. This review would also provide a reference for the safe clinical use of honey and the development of functional foods.