ObjectiveTo study in vitro antibacterial activity and physicochemical properties of three unifloral honeys (citrus, clover and cotton honeys), and to study the impacts of storage, dilution with water (33%, w/v) and autoclaving (121 °C for 15 min) on honeys characteristics. MethodsHoney samples from monofloral sources including citrus (Citrus spp.), Egyptian clover (Trifolium alexandrium) and cotton (Gossypium vitifolium) were obtained during three successive seasons (2010–2012). Physicochemical properties and antimicrobial activities of different honey samples were studies. ResultsIn honey samples stored for 12 or 24 month, colour, hydroxymethyl furfural and acidity increased, while refractive index, water activity, total soluble solids, electrical conductivity and pH remained relatively unaffected, but H2O2 values decreased. Types of honey exhibited various degrees of antibacterial activity against different indicator bacteria, wherein the highest antibacterial activity was recorded for clover honey followed by citrus and cotton honeys, respectively. Different species of bacteria were differed in their sensitivity to honey, wherein Salmonella enteritidis was the most sensitive followed by Staphylococcus aureus, Listeria monocytogenes and Escherichia coli, respectively. Storage up to 24 months at room temperature slightly reduced the antibacterial activity. The reduction levels were about 2.6% and 4.6% after 12 and 24 months, respectively. Diluting honeys with water increased the antibacterial activity by ca. 8.3%, while autoclaving decreased the antibacterial activity by ca. 13.5%. The relative contribution of the peroxide and non-peroxide components in the total antibacterial activity of fresh honeys was investigated. The antibacterial activity of honeys was mainly attributed to non-peroxide antibacterial agents, wherein their contribution was ca. 88%, while the contribution of H2O2 was only 12%. The contribution of the thermostable antibacterial components in honey was ca. 86.8%. The antibacterial activity of the fresh clover honey was compared with the effect of 16 antibiotics on indicator bacteria. Clover honey exhibited antibacterial activity comparable to that exhibited by the tested antibiotics. Moreover, antibacterial activity of water diluted fresh clover honey was higher compared to some of tested antibiotics. Clover honey appeared to deserve further investigations, since it may prove to be a promise therapeutic honey. ConclusionsHoney samples tested in this study exhibited antibacterial activity against tested pathogenic bacteria, and this activity was mainly due to non-peroxide antibacterial factors. Clover honey appeared to deserve further investigations, since it may prove to be a promising therapeutic honey.