Glacier-fed streams (GFSs) are harsh environments hosting unique, highly specialized communities. Interestingly, glaciers and their GFSs are also present in Earth’s tropical regions, where environmental characteristics contrast with GFS conditions elsewhere. Yet, despite the unique and isolated nature of tropical GFSs, little is known about their inhabitants, even though they may disappear later this century with ongoing climate change. Here, we examined diatom communities from one of the last tropical African GFSs in the Rwenzori Mountains, Uganda, to characterize the composition and diversity of this unique system. Six sediment-associated biofilm samples were collected from two reaches of a stream draining the Mt. Stanley Glacier, and the resident diatom communities were studied morphologically using light and scanning electron microscopy, as well as through the sequencing of amplicons from extracted DNA (18S and rbcL). In general, morphological results agree well with barcoding results, but each individually provides irreplaceable insights. In total, we identify 24 morphotypes utilizing light microscopy, 101 diatom Amplicon Sequence Variants (ASVs) using 18S sequences, and 65 ASVs with rbcL. Across approaches, common genera include Achnanthidium, Psammothidium, Neidium, Cymbopleura, Eunotia, and Pinnularia. However, only about half of the diversity could be assigned to the species level across methodologies, including several of the most common taxa, indicating a high level of uniqueness. Accordingly, one of the most common taxa encountered is described here as a new species, Neidium rwenzoriense sp. nov. Our results emphasize the Rwenzori Mountains as a global hotspot for endemism, and the novelty of disappearing tropical GFSs as diatom habitats.