The application range of a two-phase loop thermosyphon (TPLT) includes electronics cooling and heating and ventilation (HVAC) systems. Combining data center heat removal with HVAC systems can be beneficial in terms of reducing energy use and greenhouse gas emissions. The thermal resistance of the TPLT is the most important parameter affecting its heat transfer ability. This study presents the first experimental characteristics of the TPLT, working with novel low Global Warming Potential (GWP) fluids, including the evaporating and condensing performance. The operation of the TPLT is evaluated with pure fluids R600a, R32, and their mixture R600a/R32 at heat sink temperature in the range of 25 °C to 35 °C under heat input from 50 W to 225 W. The novel mixture presents the highest temperature at the evaporator outlet. Pure fluids R600a and R32 show the highest heat transfer coefficients and the lowest thermal resistance. The flow visualization is performed to study the boiling flow patterns. Empirical correlations are employed to predict the boiling-heat transfer coefficients. Thermal characteristics are obtained for further development of TPLT operating with environmentally friendly fluids.