Free-living amoebae FLA are ubiquitous protists. Pathogenic FLA such as N. fowleri can be found in hot springs in Guadeloupe, soil being the origin of this contamination. Herein, we analyzed the diversity and distribution of FLA in soil using a targeted metataxonomic analysis. V. vermiformis was by far the most represented species of FLA, being detected throughout the islands. Although Naegleria are mainly found in Basse-Terre region, N. fowleri was also detected in Grand Terre and Les Saintes Islands. Acanthamoeba were mainly found in areas where temperature is approx. 30 degrees C. Vannella and Vahlkampfia are randomly found in Guadeloupe islands. FLA detected in Guadeloupe include both pathogenic genera and genera that can putatively harbor microbial pathogens, therefore posing a potential threat on human health.