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Background: Microbial keratitis (MK) is a vision-threatening infection whose empiric management depends on current, location specific antimicrobial susceptibility data. No nationwide analysis has yet characterised MK in South Africa’s private healthcare sector. Aim: The study aimed to describe the microbial profile of keratitis and antimicrobial resistance patterns within the private healthcare sector in South Africa. Setting: This study was conducted within South Africa’s private healthcare sector using the nationwide laboratory system of Lancet Laboratories, a large private pathology service. Methods: This retrospective study reviewed microbiological reports of patients who underwent corneal scrape or pus swab processed by Lancet Laboratories between January 2016 and July 2024. Demographic details, microscopy results, culture isolates and antibiotic susceptibilities were extracted from laboratory reports. Results: The corneal scrapes and/or swabs of 688 patients were analysed. The overall culture positivity rate was 35.0%. Median patient age was 46 years and 55.0% were female. Gram-positive bacteria accounted for 49.4% of positive cultures (n = 132/267), gram-negative bacteria for 46.8% (n = 125/267) and fungi for 3.7% (n = 10/267). Pseudomonas aeruginosa was the predominant pathogen (29.0% of all isolates; 62.0% of gram-negative), followed by Staphylococcus epidermidis (14.0%) and Staphylococcus aureus (13.0%). Resistance rates were highest for penicillin (74.0%; n = 26/35) and chloramphenicol (27.0%; n = 36/134); gentamicin and tobramycin each showed 11.0% resistance, while ciprofloxacin and moxifloxacin exhibited 14.0% and 12.0% resistance, respectively. Contribution: This study provides private-sector, South Africa–specific microbial surveillance data for keratitis, informing empiric treatment choices and supporting judicious antibiotic use. Conclusion: The private-sector MK profile is distinguished by a high prevalence of P. aeruginosa and appreciable resistance to chloramphenicol and penicillin, whereas fluoroquinolone and aminoglycoside activity remains largely preserved. These findings support the continued use of a fluoroquinolone or fortified aminoglycoside–cephalosporin combination as empiric therapy, while underscoring the need for ongoing regional surveillance to detect emerging resistance trends.