Twenty gas samples have been collected from the natural gas manifestations of Milos Island, the majority of which is found underwater along its coast. Furthermore, three anomalous degassing fumarolic areas (Kalamos, Paleochori and Adamas) have been recognized on-land. Almost all the gases are CO2-dominated with CO2 ranging from 88 to 99% vol for the samples taken underwater, while the on-land manifestations show a wider range (15–98%) due to air contamination. Methane reaches up to 1.0% vol, H2 up to 3.2% vol and H2S up to 3.5% vol indicating a hydrothermal origin of the gases. The isotope composition of He points out to mantle contributions up to 45%, while the C-isotope composition of CO2 (from −1.9 to +1.3‰ vs. V-PDB with most of the values around −0.5‰) suggests a prevailing limestone origin. Isotope composition of CH4, ranging from −18.4 to −5.0‰ vs. V-PDB for C and from −295 to +7‰ vs. V-SMOW for H, points to a geothermal origin with sometimes evident secondary oxidation processes. Additionally, CO2-flux measurements showed high values in the three fumarolic areas (up to 1100, 1500 and 8000 g/m2/d at Kalamos, Paleochori and Adamas respectively) with the highest CO2-flux values (up to about 23,000 g/m2/d) being measured in the sea at Kanavas with a floating chamber. The south-western part of the island was covered with a lower density prospection revealing only few anomalous CO2 flux values (up to 650 g/m2/d). The total output of the island (30.5 t/d) is typical of quiescent closed-conduit volcanoes and comparable to the other volcanic/geothermal systems of the south Aegean active volcanic arc (Nisyros, Kos, Nea Kameni, Methana and Sousaki).