In this paper, we describe two new Macrobiotus species from Mississippi (USA) and Crete (Greece) by means of integrative taxonomy. Detailed morphological data from light and scanning electron microscopy, as well as molecular data (sequences of four genetic markers: 18S rRNA, 28S rRNA, ITS-2 and COI), are provided in support of the descriptions of the new species. Macrobiotus annewintersae sp. nov. from Mississippi belongs to the Macrobiotus persimilis complex (Macrobiotus clade B) and exhibits a unique egg processes morphology, similar only to Macrobiotus anemone Meyer, Domingue & Hinton, 2014, but mainly differs from that species by the presence of eyes, granulation on all legs, dentate lunulae on legs IV, and of bubble-like structures within the tentacular arms that are present on the distal portion of the egg processes. Macrobiotus rybaki sp. nov. from Crete belongs to the Macrobiotus clade A and is most similar to Macrobiotus dariae Pilato & Bertolani, 2004, Macrobiotus noemiae Roszkowska & Kaczmarek, 2019, Macrobiotus santoroi Pilato & D'Urso, 1976, and Macrobiotus serratus Bertolani, Guidi & Rebecchi, 1996, but differs from them mainly in the morphological details of its egg processes and chorion reticulation, but also by a number of morphometric characters. In light of the specific morphology of the egg processes of Macrobiotus annewintersae sp. nov. and Macrobiotus anemone, that are equipped with tentacular arms instead of proper terminal disc, we also provide an updated definition of the Macrobiotus persimilis complex.