A 3-year-old Basiliscus plumifrons developed a necrotic lesion on the tail resulting from nodules of unknown etiology. Investigation of necrotic tissue revealed several gram-negative bacteria as well as three different species of free-living amoebae. The amoebae were identified by morphological characters as belonging to the genera Acanthamoeba, Echinamoeba, and Naegleria, respectively. Partial sequencing of the 18S ribosomal gene was performed for reliable systematic determination. Two of the isolates showed thermotolerance. No isolate was growable in conventional liquid media, but the Acanthamoeba strain readily grew on a human cell line (HEp2). It remains unclear whether the amoebae fed on the coexisting bacteria or on host tissue.