Specific therapy of ocular infections often requires etiological diagnosis that is a combined effect of observation of characteristic clinical features and microbiological investigations. Clinical impression is central to guiding the laboratory investigation, and the aim of laboratory investigation is to confirm or rule out the clinical diagnosis. However, clinical features may vary considerably, and no one clinical feature may be pathognomonic of a particular pathogen. In addition, there may be a racial, geographical, and climatic difference in the distribution and type of causative agents associated with infections. Ophthalmologists have at their disposal in vivo and in vitro methods of diagnosis of ocular infections. The expertise of the clinician and the microbiologist along with the facilities available, determine the success with accurate diagnosis. A wide range of conventional and molecular techniques are available that not only provide rapid diagnosis for known common infections but have the potential to bring to the fore unknown organisms that may be associated with ocular infections.