N2-fixation is an important mechanism in microbial mats of the McMurdo Ice Shelf as nitrogen sources are limited. Here we applied molecular analyses of the N2-fixing diversity in cyanobacterial dominated microbial mats in a meltwater pond, known as Orange Pond, on the McMurdo Ice Shelf. Phylogenetic analyses of nifH genes and nifH gene transcripts were performed in association with acetylene reduction assay measurements. Eighteen phylotypes with the highest similarities to cyanobacteria, firmicutes, beta-, gamma- and deltaproteobacteria, spirochaetes and verrumicrobia were identified. All cyanobacterial nifH phylotypes grouped solely in the genus Nostoc spp. Clone-library analysis of nifH gene transcripts only identified sequences with a highest match to Nostoc spp. and acetylene reduction activity was identified in the presence of light and absence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea. These molecular results indicate that a variety of bacterial phyla possess the ability to fix nitrogen. However, under the tested conditions the only organisms actively transcribing nifH genes were Nostoc spp. This underlines the importance of Nostoc for the nitrogen budget on the McMurdo Ice Shelf.