Many seabirds including penguins are adapted to long periods of fasting, particularly during parts of the reproductive cycle and during moult. However, the influence of fasting on the gastrointestinal (GI) microbiota has not been investigated in seabirds. Therefore, the present study aimed to examine the microbial composition and diversity of the GI microbiota of fasting little (Eudyptula minor) and king penguins (Aptenodytes patagonicus) penguins during early and late moult. The results from this study indicated that there was little change in the abundance of the major phyla during moult, except for a significant increase in the level of Proteobacteria in king penguins. In king penguins the abundance of Fusobacteria increases from 1.73% during early moult to 33.6% by late moult, whilst the abundance of Proteobacteria (35.7% to 17.2%) and Bacteroidetes (19.5% to 11%) decrease from early to late moult. In little penguins, a decrease in the abundances of Firmicutes (44% to 29%) and an increase in the abundance of Bacteroidetes (11% to 20%) were observed from early to late moult respectively. The results from this study indicate that the microbial composition of both king and little penguins alters during fasting. However, it appears that the microbial composition of king penguins is more affected by fasting than little penguins with the length of fast the most probable cause for this difference.