It has been univocally shown that iron (Fe) is the primary limiting nutrient for phytoplankton metabolism in high-nutrient, low-chlorophyll (HNLC) waters, yet the question of how this trace metal affects heterotrophic microbial activity is far less understood. We investigated the role of Fe for bacterial heterotrophic production and growth at three contrasting sites in the naturally Fe-fertilized region east of the Kerguelen Islands and at one site in HNLC waters during the KEOPS2 (Kerguelen Ocean and Plateau Compared Study 2) cruise in spring 2011. We performed dark incubations of natural microbial communities amended either with iron (Fe, as FeCl3) or carbon (C, as trace-metal clean glucose), or a combination of both, and followed bacterial abundance and heterotrophic production for up to 7 days. Our results show that single and combined additions of Fe and C stimulated bulk and cell-specific bacterial production at the Fe-fertilized sites, while in HNLC waters only combined additions resulted in significant increases in these parameters. Bacterial abundance was enhanced in two out of the three experiments performed in Fe-fertilized waters but did not respond to Fe or C additions in HNLC waters. Our results provide evidence that both Fe and C are present at limiting concentrations for bacterial heterotrophic activity in the naturally fertilized region off the Kerguelen Islands in spring, while bacteria were co-limited by these elements in HNLC waters. These results shed new light on the role of Fe in bacterial heterotrophic metabolism in regions of the Southern Ocean that receive variable Fe inputs.