This study describes the new species Achnanthidium guarulhense sp. nov. from the oligo-mesotrophic Negro Lake located in a Conservation Unit in Southeast Brazil. Its morphology was analyzed through Light (LM) and Scanning Electron Microscopy (SEM). The most striking feature of the new species is its narrow linear-lanceolate axial area expanding to a linear-elliptic central area between the proximal raphe ends. This feature makes the proximal raphe ends appear more distant from each other than in other species of the genus. We also provide its ecological information and a comparison with closely related species. Our results contribute to the knowledge of the biodiversity of tropical diatoms and provide subsidies for monitoring studies.