Microbial keratitis is the most common cause of infective vision loss. The causative organism varies by region, and most cases require intensive antimicrobial therapy. The purpose of this study was to analyse the causative organisms of microbial keratitis, its presentation and economic burden from a tertiary referral hospital in Australia. A retrospective review of 160 cases of microbial keratitis was performed, over a 5-year period from 2015–2020. A wide variety of costs were considered to determine the economic burden, using standardized data from the Independent Hospital Pricing Authority and the cost of personal income loss. Our study showed the most commonly occurring pathogens were Herpes Simplex (16%), Staphylococcus aureus (15.1%) and Pseudomonas aeruginosa (14.3%). A total of 59.3% of patients were admitted, with a median length of admission of 7 days. Median cost for all presentations of microbial keratitis was AUD 8013 (USD 5447), with costs significantly increasing with admission. The total annual cost of microbial keratitis within Australia is estimated to be AUD 13.58 million (USD 9.23 million). Our findings demonstrate that microbial keratitis represents a significant economic burden for eye-related diseases and the key driving factor for the cost is the length of admission. Minimizing the duration of admission, or opting for outpatient management where appropriate, would significantly reduce the cost of treatment for microbial keratitis.