Endophthalmitis is a serious ophthalmic disease characterized by changes in the eye’s posterior segment, such as hypopyon and intraocular inflammation, vitritis being a hallmark. Infection-caused endophthalmitis can lead to irreversible vision loss, accompanied by eye pain or eye distention, and in the most severe cases the removal of the eyeball. Microorganisms such as bacteria, fungi, viruses, and parasites typically account for the disease and the entry pathways of the microbial can be divided into either endogenous or exogenous approaches, according to the origin of the etiological agents. Exogenous endophthalmitis can be derived from various occasions (such as post-operative complications or trauma) while endogenous endophthalmitis results from the bloodstream which carries pathogens to the eye. This review aims to summarize the application of new technology in pathogen identification of endophthalmitis so as to prevent the disease and better guide clinical diagnosis and treatment.