The Southern Ocean is largely a High Nutrient Low Chlorophyll (HNLC) region where macronutrient concentrations are high and phytoplankton productivity is low. However, there are productive ‘hot spots’ that sustain large phytoplankton blooms. These areas, maintained by natural iron (Fe) fertilization, are important for the Southern Ocean ecosystem and for driving carbon export. Fe addition on-deck bioassay experiments were conducted on two cruises to the Scotia Sea region of the Southern Ocean (austral spring 2006 and summer 2008) to better understand how Fe controls the microplankton (20–200 μm) community structure on a seasonal basis. Light microscopy and fast-repetition rate fluorometry were used to examine changes in the species composition and physiological status of the microplankton community. Bioassays were carried out in three contrasting regions of the Scotia Sea: (1) a naturally Fe-fertilized, high chlorophyll area downstream (north and northwest) of the Islands of South Georgia (DSG); (2) a low Fe, low chlorophyll area upstream (south) of the Islands of South Georgia (USG); and (3) a naturally Fe-fertilized area north of the South Orkney Islands (SOI). Multivariate statistics were applied to the light microscopy results, showing significant differences between the initial microplankton communities for each of the bioassays. These differences were primarily spatial (between regions) and secondarily temporal (between seasons). Significant microplankton community shifts occurred in three of five bioassays, those in spring and summer USG and in summer DSG only. In summer, USG community responses increased significantly in medium (100–1000 pg C cell−1) and large (>1000 pg C cell−1) diatom species in response to Fe addition. Such a response was consistent with relief from in situ Fe limitation, which favours larger microplankton species with higher Fe requirements and subject to lower grazing pressures. The largest biomass increase in Fe-treated bioassay bottles was in Pseudonitzschia spp., which suggests that this genus may be a particularly important member of the microplankton community in the Scotia Sea.