Ocular infection is caused by both endogenous (resident) and exogenous (environmental) microbes. As the ocular surface interacts with both outer environment and its own resident microbiota, clinical ocular samples are predicted to contain a diverse set of microorganisms. Microscopy of sample smears is an important step in the diagnostic process of infectious diseases to interpret the culture results. Traditional culture techniques have several limitations in the detection and/or identification of uncharacterized bacteria of environmental origin. Molecular biological techniques, such as polymerase chain reaction of pathogen-specific virulence genes, 16S rRNA gene clone library analysis, and next-generation sequencing of 16S rDNA amplicons, compensate for diagnostic culture techniques in diagnosing infectious diseases. These techniques are expected to provide novel insights into the ocular microbiota and pathology of ocular infections. In this article, we describe various ocular infections, including contact lens-related keratitis, silicone buckle infection, and dacryocystitis, which were analyzed using molecular biological techniques. The advantages and disadvantages of these highly sensitive and inclusive microbiological detection systems for ocular infections are discussed.