Shallow lakes are common across the Arctic landscape and their ecosystem productivity is often dominated by benthic, cyanobacterial biofilms. Many of these water bodies freeze to the bottom and are biologically inactive during winter, but full freeze-up is becoming less common with Arctic warming. Here we analyzed the microbiome structure of newly discovered biofilms at the deepest site of a perennially ice-covered High Arctic lake as a model of polar microbial communities that remain unfrozen throughout the year. Biofilms were also sampled from the lake's shallow moat region that melts out and refreezes to the bottom annually. Using high throughput small subunit ribosomal RNA sequencing, we found more taxonomic richness in Bacteria, Archaea and microbial eukaryotes in the perennially unfrozen biofilms compared to moat communities. The deep communities contained both aerobic and anaerobic taxa including denitrifiers, sulfate reducers, and methanogenic Archaea. The water overlying the deep biofilms was well oxygenated in mid-summer but almost devoid of oxygen in spring, indicating anoxia during winter. Seasonally alternating oxic-anoxic regimes may become increasingly widespread in polar biofilms as fewer lakes and ponds freeze to the bottom, favoring prolonged anaerobic metabolism and greenhouse gas production during winter darkness.