Two new species of Paracalanidae, Giesbrecht, 1893, have been described. Paracalanus brasiliensis sp. nov. and Bestiolina brasiliensis sp. nov. were registered in four estuaries on the central coast of Brazil. Paracalanus brasiliensis sp. nov. females differ from their congeners mainly with regard to body size, the structure of swimming legs 1–4, absence of bristles in the coxopodites, spinules between the spines in the third segment of the exopodite, and the shape of the seminal receptacles. The uniformity of the number of spinules and their location on the anterior face of the second exopodite of legs 2–3 and the absence of spinules on the endopodite of legs 3–4 differentiate Bestiolina brasiliensis sp. nov. females from other Bestiolina Andronov, 1991, species. In the males of both species, the main diagnostic features (swimming leg seta, spine formula, and ornamentation) are generally observed in females with a few additional characteristics. Genetic divergence analyses based on partial mitochondrial COI (mtCOI) sequences revealed no genetic divergence between Paracalanus brasiliensis sp. nov. and Paracalanus sp. E. sensu Cornils and Held (2014), demonstrating that they are mutually conspecific. mtCOI sequence data from Bestiolina brasiliensis sp. nov. identified a clade with high bootstrap support that separated the specimens in this study from other Bestiolina species. The present report provides the first morphological description of females and males of both Paracalanus brasiliensis sp. nov. and Bestiolina brasiliensis sp. nov. and presents molecular evidence for species specificity. Matters regarding the validity of these species are also discussed.