Abstract: To evaluate the physio-chemical properties and antibacterial activities of A. andreniformis honey (AAH), its physio-chemical properties were determined via AOAC and other methods. The botanical sources and antibacterial activity against bacteria of S. aureus, E. coli and C. Violaceum were determined by using DNA metabarcoding and the agar well diffusion methods, respectively. Results: The three AAH samples harvested at the same time and location had significant differences in physio-chemical characters, there were significant differences in water, ash,pH, acid, free acid, lactone acid, glucose, fructose, amylase activity, 5-hydroxymethylfurfural contents, conductivity among these three honey samples. There were little significant differences in maltose and sucrose contents. There were significant differences among the types and number of botanical sources in these samples; there was no common plant source among the three honey samples. There was only one common plant source of Crassocephalum rubens in AAH1 and AAH2, with gene abundance of 16.16% and 4.15%, respectively. There was only one common plant source of Flueggea virosa in AAH1and AAH3, with gene abundance of 2.06% and 31.32%, respectively. The antibacterial activity of AAH3 against S. aureus was the strongest and better than that of Manuka honey. The antibacterial activity of AAH3 against E. coli was the same as that of MH1 honey and was better than other samples. The antibacterial activity of AAH1 against C. Violaceum was the strongest, but weaker than that of MH3 and MH1 honey. Conclusion: A. andreniformis honey has a good antibacterial activity. The results can provide a theoretical basis for the development and utilization of A. andreniformis honey.