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To report the co-isolation incidence of Acanthamoeba and Vahlkampfia in amoebic keratitis from a tertiary care institution in Madrid, Spain.In this retrospective case series, 7 eyes of 7 consecutive patients with culture-proven or polymerase chain reaction–positive Acanthamoeba keratitis were seen at a tertiary care institution from January 2010 to April 2011, and their charts were reviewed.Two of 7 patients showed mixed Acanthamoeba and Vahlkampfia keratitis. Good clinical response to the treatment was strongly correlated with early diagnosis, whereas delayed diagnosis resulted in poor response to the treatment in single or mixed infection.Co-isolation of Vahlkampfia and Acanthamoeba in Acanthamoeba-like keratitis has recently been detected in our population. This finding should raise awareness of the existence of other amoeba different from Acanthamoeba causing keratitis. There are not enough cases yet to determine the impact of mixed amoebic keratitis in the prognosis of this disease.