The tandem scanning confocal microscope (TSM) was adapted for in vivo examination of the cornea in rabbits with experimental bacterial and fungal keratitis. Compared to slit lamp biomicroscopy, the TSM provides superior lateral and axial resolution and serial optical sectioning capability, which may be useful for identification of corneal pathogens in the early stages of infection. We used the TSM to examine normal rabbit eyes infected with bacteria (Bacillus cereus) and a filamentous fungus (Aspergillus). We also examined a human cornea removed by penetrating keratoplasty after a clinical diagnosis of amoebic keratitis. In the early stages of bacterial infection, slit lamp examination revealed a nonspecific minimal stromal haze and limbal injection indistinguishable from sterile ulcers and epithelial defects. With the TSM, bacteria were visible as highly refractile bodies in the epithelium and superficial stroma. Branching fungal hyphae were also easily identified by the TSM, as were Acanthamoeba cysts and parasites in the subepithelial stroma. Our results indicate that this technique may provide a new modality for quickly and accurately identifying the agent of corneal infection, thereby facilitating prompt and appropriate treatment.