Ecuadorian honey samples of eight genera and 12 stingless bee species from three geographic regions (Andes, Amazon and Pacific) were studied for their physicochemical parameters, chemical composition, mineral elements and antimicrobial activity. Meliponine honey is acidic and has a high water content, but our study revealed substantial chemical variation. Oxytrigona mellaria was highest in proteins, while Melipona sp. had the most free amino acid content. Five species of honey contained vitamin C, which was highest in O. mellaria. The most abundant minerals were the macronutrients, potassium and calcium. All honey inhibited microbial growth in gram-negative and gram-positive multiresistant human pathogens, with O. mellaria and Trigona silvestriana being most effective against both bacteria.