Honeybee (Apis mellifera L.) workers act as passive vectors of Paenibacillus larvae spores, which cause the quarantine disease American foulbrood (AFB). We assessed the relative proportions of P. larvae within the honeybee microbiome using metabarcoding analysis of the 16 S rRNA gene. The microbiome was analyzed in workers outside of the AFB zone (control - AFB0), in workers from asymptomatic colonies in an AFB apiary (AFB1), and in workers from colonies exhibiting clinical AFB symptoms (AFB2). The microbiome was processed for the entire community and for a cut-off microbiome comprising pathogenic/environmental bacteria following the removal of core bacterial sequences; varroosis levels were considered in the statistical analysis. No correlation was observed between AFB status and varroosis level, but AFB influenced the worker bee bacterial community, primarily the pathogenic/environmental bacteria. There was no significant difference in the relative abundance of P. larvae between the AFB1 and AFB0 colonies, but we did observe a 9-fold increase in P. larvae abundance in AFB2 relative to the abundance in AFB1. The relative sequence numbers of Citrobacter freundii and Hafnia alvei were higher in AFB2 and AFB1 than in AFB0, whereas Enterococcus faecalis, Klebsiella oxytoca, Spiroplasma melliferum and Morganella morganii were more abundant in AFB0 and AFB1 than in AFB2.