The study aims to compare the direct polymerase chain reaction with microbial culture for the detection and fungal pathogens in infectious keratitis. A total of 81 corneal ulcers were culture and analyzed prospectively. PCR was performed with all corneal scrapping with fungal and bacteria specific primers. PCR products were analysed and compared with the culture results using standard methods. Of the 81 samples, 80 were positive by PCR, 51 for fungi and 29 for bacteria. Out of 51 PCR positive samples, 22 samples were culture positive and 29 were culture negative. The majority of PCR genotyped samples matched the positive culture results. The positive detection rate of 80/81 (98.8%) with high suspicion of fungal keratitis and positive detection rate of direct PCR 50/51(98.0%) were observed. The sensitivities for the diagnosis of fungal keratitis with direct PCR and culture were 98.0% (50/51) and 43.1% (22/51) (p< 0.001) whereas the specificities were 100.0% (2/2) and 100.0% (1/1) respectively. The time required to complete the direct PCR was only 3 hours. The direct PCR assay is a rapid diagnostic technique with high sensitivity and specificity for infectious keratitis and it is expected to have impact on the diagnosis and treatment of infectious keratitis.