Adults of the dusk-flying and rotting wood-nesting bees Megalopta genalis and/or M. ecuadoria were collected at Barro Colorado Island, The Smithsonian Tropical Research Institute (BCI, STRI), Panama and La Selva, Costa Rica, and examined for nematodes. Nematodes were recovered as dauer juveniles from the Dufour's gland of female bees and aedeagus of male bees. Adult nematodes isolated from M. genalis (BCI and La Selva) and M. ecuadoria (BCI) were successfully cultured and kept in the laboratory. All three populations were used for morphological observations and molecular analyses and were identified as a new species that is described herein as Acrostichus megaloptae n. sp. It is characterised by its stomatal morphology, possession of six triangular cuticle flaps covering stomatal opening, duplicated cheilo- and gymnostomatal walls, large dorsal tooth and sclerotised ventral stegostomatal ridges, female with vulval flap, male spicule and gubernaculum morphology, i.e., relatively straight spicule with oval-shaped manubrium, rounded flap-like rostrum and separated and strongly ventrally curved spicule with bifurcate tip, gubernaculum L-shaped in lateral view and anchor-shaped in ventral view, and filiform tail of both sexes. Sequences from the three A. megaloptae n. sp. geographical/host isolates were not significantly different and molecular phylogenetic analysis and biological and morphological comparisons place the new species close to A. halicti from Halictus ligatus and A. puri from Augochlora pura from North America, although the new species is distinguished from A. halicti and A. puri based upon its stomatal morphology of males and females, male spicule and gubernaculum morphology and female vulval structure.