As part of the All Taxa Biodiversity Inventory (http://www.dlia.org), we are conducting a large-scale multihabitat inventory of tardigrades in the Great Smoky Mountains National Park (GSMNP) in Tennessee and North Carolina, USA. Here we report our findings for the genus Macrobiotus (Eutardigrada, Macrobiotidae). Two new species, Macrobiotus martini sp. nov. and Macrobiotus halei sp. nov., are described from moss, lichen, soil and leaf litter samples. Macrobiotus martini sp. nov. differs from all other species of the Macrobiotus hufelandi group by having a very simple buccal armature without bands of teeth, very large elliptical cuticular pores, unique characteristics of the egg, and other morphometric characters. Macrobiotus halei sp. nov. differs from other species in the Macrobiotus richtersi group by having very small cuticular tubercles, well-developed macroplacoids, unique characteristics of the egg, and other morphometric characters. Seven additional Macrobiotus species have been identified in the GSMNP: M. harmsworthi Murray, 1907, M. hufelandi Schultze, 1834, M. islandicus Richters, 1904, M, montanus Murray, 1910, M. pallarii Maucci, 1954, M. recens Cuénot, 1932, and M. tonollii Ramazzotti, 1956. All of these except M. harmsworthi are new records for the national park. In addition, M. pallarii is a new record for North Carolina, M. islandicus is a new record for North Carolina and Tennessee, M. montanus is a new record for the eastern USA, and M. recens is a new record for the USA.