Fungal keratitis can lead to pain and impaired vision. Current treatment options include antifungal agents and therapeutic penetrating keratoplasty. An emerging option for the management of keratitis is photodynamic antimicrobial therapy (PDAT) which uses a photosensitizer rose bengal activated with green light. Utilizing a pulsed irradiation, rather than the standard continuous irradiation may have a similar antimicrobial effect with less total energy. This study is to compare pulsed and continuous rose bengal mediated PDAT for inhibition of six fungal isolates on agar plates: Fusarium solani, Fusarium keratoplasticum, Aspergillus fumigatus, Candida albicans, Paecilomyces variotti, and Pseudoallescheria boydii. Isolates were mixed with 0.1% rose bengal and exposed to three irradiation conditions: (1) 30-minute continuous (10.8J/cm2), (2) 15-minute continuous (5.4J/cm2), (3) 30-minute pulsed (5.4J/cm2). Plates were photographed at 72 hours and analyzed with custom software. At 72 hours, 30-minute continuous rose bengal mediated PDAT inhibited all six fungal species. Fungal inhibition was analogous between 30-minute continuous and 30-minute pulsed test groups, with the exception of A. fumigatus. The 15-minute continuous irradiation was less effective when compared to both 30-minute continuous and 30-minute pulsed groups. These in vitro results demonstrate the potential strength of pulsed rose bengal mediated PDAT as an adjunct treatment modality for fungal keratitis.