Massive amounts of organic carbon have accumulated in Arctic permafrost and soils due to freezing temperatures and saturating water that creates anoxic conditions and limits aerobic microbial respiration. Alternative electron acceptors are thus required for microbes to oxidize and degrade organic carbon in these soils. Iron or iron oxides have been recognized to play an important role in carbon cycle processes in Arctic soils, although its exact form and role as an electron acceptor or donor remain poorly known, particularly in the tundra surface water where iron reduction and oxidation can occur concurrently. Here, Arctic films, commonly observed as opaque, blue sheens from tundra surface waters on the Seward Peninsula of Alaska, were characterized with a suite of microscopic and spectroscopic methods. Energy dispersive X-ray and electron energy loss spectroscopic analyses revealed that the major components of the films is mixed-valence iron oxide magnetite (Fe2+Fe23+O4) nanoparticle (< 5 nm) aggregates. These nanoparticles were associated with extracellular polymeric substances (polysaccharides and proteins) evidenced by Fourier transform infrared and surface enhanced Raman scattering spectroscopies. The formation of magnetite nanoparticles associated with organic filaments at the air–water interface likely represents an important mechanism of regenerating electron acceptors for continual anaerobic microbial respiration of organic carbon within poorly drained Arctic tundra.