We analyzed bacterial and fungal contamination within the contact lens care systems of ten patients who had Acanthamoeba detected within their care systems. Seven patients had Acanthamoeba keratitis, one had Pseudomonas keratitis, and the remaining two were asymptomatic. Gram-negative bacteria were found in all ten care systems, and Pseudomonas was found in six. Bacillus species, the only gram-positive bacteria isolated, were found in five systems. Fungi were isolated in six care systems. The use of homemade saline and the two-cup method of peroxide disinfection were associated with microbial contamination. Acanthamoeba organisms were found only in contact lens cases or solutions that also had bacterial and in many cases fungal contamination, suggesting that the presence of bacterial and fungal contamination within the contact lens care system may be an important element for the survival and growth of Acanthamoeba.