The most well-known functional properties of honey are its antioxidant and antimicrobial activities. The bioactive components of honey are afected by the lora from which it is produced and by geographical variations. Phenolic compounds promote, among other activities, high antioxidant action, being capable of minimizing intracellular oxidative damage associated with cellular aging, apoptosis and neurodegenerative diseases. A living cell system would provide a beter platform for determining antioxidant activity, since the bioactive honey compounds can act modulating antioxidant defense gene expression. Indeed, phenolic compounds, amino acids and reducing sugars are among the substances responsible for honey antioxidant activity. Most of phenolic compounds also exert antimicrobial activity against a number of pathogens and spoilage microorganisms. The antimicrobial activity of honey is also due to the action of enzymes. In addition, honey was found to contain lactic acid bacteria (LAB), which itself produce a myriad of active compounds that remain in variable amounts in mature honey. In addition, these antioxidant compounds might play a key role as prebiotic, protecting and stimulating growth of probiotic bacteria. Oligosaccharides present in honey are well-known prebiotic substances stimulating growth, activity and protecting probiotic bacteria during passage through the gastrointestinal tract and during storage of the products. This chapter describes the main bioactive components of honey, especially with respect to the phenolic compounds and their antioxidant activity and assay methods.