Background: Microbial keratitis (MK) is a major cause of blindness worldwide. Few studies have assessed the HRT3 in vivo confocal microscope (IVCM) in the diagnosis or management of MK. This PhD aimed to investigate these questions and was based in a high incidence setting for MK: Aravind Eye Hospital, Madurai, India. Methods: A prospective observational study of 252 severe MK patients was conducted with follow-up at days 7, 14 and 21 post-enrolment. Quantitative PCR of corneal swabs obtained at presentation validated transcriptome results. IVCM images were evaluated in this cohort for diagnostic accuracy, and ability to monitor outcome. Results: For fungal detection, HRT3 IVCM had a high sensitivity (85.7%; 95% CI: 82.2%– 88.6%) and specificity (81.4%; 95% CI: 76.0%–85.9%). For Acanthamoeba, the sensitivity was 88.2% (95% CI: 76.2%–94.6%) and specificity was 98.2% (95% CI: 94.9%–99.3%). Mean fungal branching angle in IVCM images was not significantly different for Fusarium sp. (59.7°; 95% CI: 57.7°–61.8°) versus Aspergillus sp. (63.3°; 95% CI: 60.8°–65.8°; p=0.07). At presentation, anterior corneal IVCM morphology associated with BK included bullae (OR 9.99, 95% CI: 3.11–32.06, p<0.001), and in FK a honeycomb distribution of inflammatory cells (OR 2.74, 95%CI: 1.01–7.40, p=0.047). Poor outcomes in FK were associated with stellate interconnected cellular processes with no visible nuclei (OR 2.28, 95% CI: 1.03-5.06, p=0.043) in baseline IVCM images, and fungal filaments (OR 6.48, 95% CI:2.50-16.78, p<0.001) or inflammatory cells in a honeycomb distribution (OR 5.24, 95% CI: 1.44-19.06, p=0.012) in final visit images. Conclusions: HRT3 IVCM can yield a high diagnostic accuracy. Fungal branching angle in IVCM images does not differentiate between Fusarium and Aspergillus keratitis. IVCM image morphologies may be associated with causative organism or clinical outcome in MK.