Importance Antimicrobial resistance data from bacterial keratitis in Australia are lacking. Background Antimicrobial resistance is a global health threat. Bacterial keratitis is an ophthalmic emergency requiring immediate and effective treatment. Design Retrospective cohort study of bacterial isolates and antibiotic susceptibility profiles at a quaternary hospital in Sydney, Australia. Participants Two hundred and twenty-four corneal scrapes from patients from January 1 to December 31, 2016. Methods Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry identified bacteria. The Calibrated Dichotomous Sensitivity (CDS) method determined antibiotic susceptibilities. Main outcome measures Isolated organisms and antibiotic susceptibilities. Results One hundred and sixty-eight scrapes of 224 (75%) were culture positive. One hundred and thirty-one patients had a single organism isolated and 21 had mixed bacterial growth. Of the 157 organisms isolated, 131 (83%) were Gram-positive and 27 (17%) Gram-negative. Of the Gram-positive organisms, 75 (57%) were coagulase-negative Staphylococci (CoNS), 15 (11%) Staphylococcus aureus (including one methicillin-resistant Staphylococcus aureus [MRSA]) and 8 (6%) Corynebacterium spp. Of the Gram-negative organisms, 15 (58%) were Pseudomonas aeruginosa. With methicillin-sensitive Staphylococcus aureus (MSSA) resistance to chloramphenicol was 21%, ciprofloxacin 7% and gentamicin 7%. With CoNS resistance to cefalotin was 9%, gentamicin 9% and ciprofloxacin 9%. With Corynebacterium spp. resistance was 40% to cefalotin, chloramphenicol 25% and ciprofloxacin 14%. Conclusions and relevance Staphyloccocus spp. and Pseudomonas spp. were the most common microorganisms isolated. There was low resistance to cefalotin and ciprofloxacin for these isolates. More than 90% of these would be covered by current therapeutic recommendations for empiric therapy in Australia.