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Free-living amoebae (FLA) are ubiquitous protists. Pathogenic FLA such as <i>N. fowleri</i> 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. Soil samples (n = 107) were collected from 40 sites. DNA was extracted directly from soil samples or from FLA cultivated at different temperatures (30, 37 and 44 °C). Metabarcoding studies were then conducted through FLA 18SrDNA amplicons sequencing; amplicon sequence variants (ASV) were extracted from each sample and taxonomy assigned against SILVA database using QIIME2 and SHAMAN pipelines. <i>Vermamoeba</i> were detected in DNA extracted directly from the soil, but to detect other FLA an amoebal enrichment step was necessary. <i>V. vermiformis</i> was by far the most represented species of FLA, being detected throughout the islands. Although <i>Naegleria</i> were mainly found in Basse-Terre region, <i>N. fowleri</i> was also detected in Grand Terre and Les Saintes Islands. <i>Acanthamoeba</i> were mainly found in areas where temperature is approx. 30 °C. <i>Vannella</i> and <i>Vahlkampfia</i> were 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 to human health.