Free-living amoebae (FLA) thrive in diverse environmental conditions. The present study aimed to define the FLA distribution from the Caspian Sea as well as from hospital ward dust from Guilan, Iran. Seawater (20) and hospital ward dust samples (100) were collected from May to June 2018. Seawater samples were vacuum filtered through a 0.45 μm pore-size membrane. Dust was collected using sterile gauze, washed with sterile distilled water, with washings collected thereafter. Washings were similarly filtered as seawater samples. FLA from the filtered material was cultivated in non-nutrient agar. Molecular analysis was performed by PCR and sequencing using specific primers for Acanthamoeba, Naegleria, and Vermamoeba/Hartmanella. Culture and PCR returned 50 and 65% positivity, respectively, for seawater samples where sequencing revealed Acanthamoeba T2, T5 and T6 genotypes and A. palestinensis and A. lenticulata, as well as N. dobsoni and N. clarki. In addition, 30% amoebic growth and 16% PCR detection were observed from hospital ward dust samples where sequencing revealed Acanthamoeba T2, T4 and T11 genotypes and A. castellanii, A. palestinensis and A. stevensoni as well as N. clarki. For both seawater and dust samples, Acanthamoeba was the dominant isolate. The detection of potentially pathogenic FLA from seawater may pose a threat to the public, while the presence of the same in dust spells threats to both hospital staff and patients, in particular, immunocompromised individuals. Public education, awareness, improved sanitation and hygiene, and the crafting of diagnostic strategies for the early detection of FLA in humans are necessary for the mitigation and management of potential human infection cases.