ABSTRACT.- Insect pollinators are threatened worldwide, being the exposure to multiple pesticides one of the most important stressor. The herbicide Glyphosate and the insecticide Imidacloprid are among the most used pesticides worldwide, although different studies evidenced their detrimental effects on non-target organisms. The emergence of glyphosate-resistant weeds and the recent ban of imidacloprid in Europe due to safety concerns, has prompted their replacement by new molecules, such as glufosinate-ammonium (GA) and sulfoxaflor (S). GA is a broad-spectrum and non-selective herbicide that inhibits a key enzyme in the metabolism of nitrogen, causing accumulation of lethal levels of ammonia; while sulfoxaflor is an agonist at insect nicotinic acetylcholine receptors (nAChRs) and generates excitatory responses including tremors, paralysis and mortality. Although those molecules are being increasingly used for crop protection, little is known about their effects on non-target organisms. In this study we assessed the impact of chronic and acute exposure to sublethal doses of GA and S on honey bee gut microbiota, immunity and survival. We found GA significantly reduced the number of gut bacteria, and decreased the expression of glucose oxidase, a marker of social immunity. On the other hand, S significantly increased the number of gut bacteria altering the microbiota composition, decreased the expression of lysozyme and increased the expression of hymenoptaecin. These alterations in gut microbiota and immunocompetence may lead to an increased susceptibility to pathogens. Finally, both pesticides shortened honey bee survival and increased the risk of death. Those results evidence the negative impact of GA and S on honey bees, even at single exposition to a low dose, and provide useful information to the understanding of pollinators decline. © 2022