Abstract Honeybee ( Apis mellifera ) ingestion of toxic nectar plants can threaten their health and survival. However, little is known about how to help honeybees mitigate the effects of toxic nectar plant poisoning. We exposed honeybees to different concentrations of Bidens pilosa flower extracts and found that B. pilosa exposure significantly reduced honeybee survival in a dose‐dependent manner. By measuring changes in detoxification and antioxidant enzymes and the gut microbiome, we found that superoxide dismutase, glutathione‐S‐transferase and carboxylesterase activities were significantly activated with increasing concentrations of B. pilosa and that different concentrations of B. pilosa exposure changed the structure of the honeybee gut microbiome, causing a significant reduction in the abundance of Bartonella ( p < 0.001) and an increase in Lactobacillus . Importantly, by using Germ‐Free bees, we found that colonization by the gut microbes Bartonella apis and Apilactobacillus kunkeei (original classification as Lactobacillus kunkeei ) significantly increased the resistance of honeybees to B. pilosa and significantly upregulated bee‐associated immune genes. These results suggest that honeybee detoxification systems possess a level of resistance to the toxic nectar plant B. pilosa and that the gut microbes B. apis and A. kunkeei may augment resistance to B. pilosa stress by improving host immunity.