Honey bee are immensely important pollinators, but their numbers are in decline due to both known pathogens, such as Nosema ceranae and Varroa destructor, and the yet not fully understood phenomenon called Colony Collapse Disorder. The overwintering process is straining for the hive and is a time where many hives are lost, therefore it is of importance to understand how to best care for the colonies during this time. This study was performed to try to determine how the composition of the intestinal microbiome of the Buckfast hybrid of the honey bee, Apis mellifera, living in Sweden, changes from autumn to during overwintering and if the diet affects the composition of the intestinal microbiome during overwintering. The composition of the intestinal microflora was investigated through Nanopore sequencing of the 16S rRNA gene. The data clearly indicates a shift in the composition of the intestinal microbiome from autumn to during overwintering in the form of declining abundance of the genera Lactobacillus, Bombilactobacillus, Gilliamella and Frischella, and an increasing abundance of the Bartonella genus. The data also clearly reveals that diet does affects the composition of the intestinal microbiome, with the group being fed a substituted diet of sugar-water having more species of and a larger abundance of opportunistic bacterial colonizers compared to the group being fed a natural diet of the colonies own produced honey. The data also shows that the individuals within the group being fed sugar were less homogenous than those that were fed honey.