The bacterium Paenibacillus larvae and the mite Varroa destructor are two of the most severe biotic stressors affecting honeybees and are responsible for American foulbrood and varroosis respectively. To control these pathogens, beekeepers regularly apply synthetic acaricides or antibiotics to parasitized hives. However, antibiotic and acaricide overuse over time leads to resistance in bacteria strains and mite populations respectively, not to mention the residual contamination of bee products with these chemicals. The development of alternative and effective control methods of bee diseases is therefore crucial. In recent years, natural substances from plant extracts have emerged as the basis of suitable control methods to treat bee colonies parasitized by both P. larvae and V. destructor . Our aim was to evaluate the bioactivity of ethanolic and methanolic hop leaf extract (species: Humulus lupulus L, varieties: Victoria, Spalt, and Cascade) against P. larvae , V. destructor , and A. mellifera . The bactericidal activity against P. larvae was evaluated by the broth microdilution method. Topical administration protocols were used to determine the bioactivity of hop extracts on V. destructor and A. mellifera . Total polyphenols, flavonoids, saponins, and antioxidant capacity were determined for each hop leaf extract tested. The Victoria extract had the highest concentration of phenolic compounds, whereas Cascade and Victoria extracts had higher concentrations of the glycoside saponin. All hop extracts presented low toxicity against A. mellifera bees after 48 h of topical administration (except for Cascade ethanolic extract which reached a maximum of 36% of bee mortality). Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values ranged from 0.69 to 2.75 mg/kg for the Cascade variety, 1.38 to 5.5 mg/kg for the Spalt variety, and 5.5 to 11 mg/kg for the Victoria variety. After 48 h, the acaricidal activity for the ethanolic extract of the Victoria variety reached a value close to 80%, while the methanolic extract of Cascade showed an acaricidal activity close to 70%. The results reported in this study support the potential use of methanolic and ethanolic extracts of hop leaves from Argentina as promising natural alternatives for varroosis and American foulbrood control.Paenibacillus larvae / Varroa destructor / Apis mellifera / natural extracts / Humulus lupulus / chemical composition