In this study, chemical composition, physicochemical properties and bioactivity of two essential oils of Rosmarinus officinalis extracted from plant material with different drying treatments against Apis mellifera, Varroa destructor and Paenibacillus larvae were assessed. The lethal concentration 50 (LC50) for mites and bees was estimated using a complete exposure method test. The broth microdilution method was followed in order to determine the minimum inhibitory concentrations (MICs) of the essential oils against P. larvae. Physicochemical properties were similar in both the essential oils, but the percentage of components showed certain differences according to their drying treatment. β-Myrcene and 1,8-cineole were the main constituents in the oils. The LC50 for complete exposure method at 24, 48 and 72 h was minor for mites exposed to R. officinalis essential oil dried in oven conditions. MIC values were 700-800 µg mL(-1) and 1200 µg mL(-1) for R. officinalis dried in air and oven conditions, respectively. The results reported in this research show that oil toxicity against V. destructor and P. larvae differed depending on the drying treatment of the plant material before the distillation of essential oil.