Despite concern around plastic pollution in subtropical gyres, our understanding of the biological component of the neustonic layer (upper 20 cm of the ocean) in these areas is poor. Here we investigate the neuston (excluding copepods) assemblages across the Southern Indian Subtropical Gyre using triplicate samples collected by manta-trawls from 28 stations along a transect at latitude 20 S during June/July 2015. The vertical structure of the water column at each station was assessed using a CTD. With the exception of siphonophores, all non-copepod neuston were identified to morphospecies. Assemblages were dominated by ostracods, mysids, larval chaetognaths and siphonophores. The majority of collected specimens comprised facultative neuston, which were more common at night than during the day. Neuston assemblages from the east were quite distinct from the balance of samples, and their distribution reflected that of the warm, low salinity water associated with the Indonesian Throughflow. Two anticyclonic eddies had been documented at the time of the survey between 70 and 85° E, and this area was associated with a distinct neuston assemblage of generally low abundance. The key environmental factors that were linked to assemblage structure were associated with longitude - salinity, fluorescence and temperature at 200 m, emphasising the interaction between Indonesian Throughflow Water and the South Equatorial Current, eddy-related process and settlement of meroplankton. The study highlights the value of using morphospecies in studies of plankton assemblages.