To describe the epidemiological and laboratory characteristics of microbial keratitis at a referral center in Brazil. Charts of all patients referred to the Ocular Microbiology Laboratory at Federal University of São Paulo (UNIFESP) from July 1975 to September 2007 were retrospectively reviewed. The following data were recorded: age, gender, involved eye, use of ocular medication, previous trauma or surgery, contact lens wear and the results of laboratory cultures. The study included 6,804 corneal cultures. The mean age was 42.1 ± 21.4 years. The male-to-female ratio was 1.5:1. Positive cultures were obtained in 3,309 (48.6%) cases. Of these, bacteria were isolated in 2,699 (39.7%), fungi in 364 (5.3%) and Acanthamoeba in 246 (3.6%) samples. Positive bacterial cultures were 2.7-fold more frequent in patients with previous use of steroids (P < 0.01), and a 30% reduction in positive bacterial cultures was observed in patients with previous use of antibiotics (P < 0.01). A total of 1,524 patients (22.4%) had a past history of ocular surgery. Contact lens wearers showed a 1.7 times greater chance of having an Acanthamoeba-positive culture (P < 0.01). Previous ocular trauma was present in 1,118 (16.4%) cases and injury caused by plants showed a 3.8 times greater chance of a positive fungal culture (P < 0.01). Bacterial organisms were identified as the most frequent agent followed by fungi and Acanthamoeba. Prescription of steroids and antibiotics prior to corneal scrapings may modify the laboratory test results. Previous corneal surgery, contact lens wear and ocular trauma have been shown to be risk factors for bacterial, Acanthamoeba and fungal keratitis, respectively.