To analyze epidemiological and microbiological aspects of microbial keratitis in children and adolescents. This retrospective cohort study was conducted at the Department of Ophthalmology and Visual Science, Escola Paulista de Medicina, Universidade Federal de São Paulo, between July 15, 1975, and December 31, 2010. We analyzed corneal samples from 859 patients with clinical suspicion of infectious keratitis, comparing epidemiological and microbiological characteristics of bacterial keratitis with those of non-bacterial and non-viral keratitis. We also compared Gram-positive and Gram-negative pathogens in patients with bacterial keratitis. We created a susceptibility profile of the bacterial microorganisms studied. Of the 859 patients, 346 (40.3%) showed positive culture results for non-viral microorganisms. Teenagers (13-18 years) made up the group with the highest number of patients with keratitis (164, 47.4%). The most frequent risk factors for keratitis were trauma (33.5%) and previous ocular surgery (24.9%). Gram-positive bacteria (71.8%) were the most often isolated, with coagulase-negative Staphylococcus (23.8%) the most prevalent microorganism. Logistic regression analysis showed age (p=0.002), topical antimicrobial drug use (p=0.01), and trauma due to non-chemical burns (p=0.005) were risk factors for non-bacterial keratitis. Age (p=0.01) was also a risk factor for Gram-negative bacterial keratitis. Our study showed that in the age range studied, the prevalence of keratitis caused by Gram-negative bacteria or by the non-viral microorganisms evaluated increases with age. Previous use of topical antimicrobial drug and trauma due to non-chemical burns are associated with non-bacterial keratitis. Knowledge of the risk factors and the microorganisms involved may help improve treatment of keratitis in children and adolescents and minimize visual impairment.