We evaluated the polyphenol content and the α-glucosidase activity exhibited by different monofloral honeys of Italian origin. Their capacity to act on different pathogenic (Acinetobacter baumannii, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, and Staphylococcus aureus) as well as probiotic bacteria (Lacticaseibacillus casei, Lactobacillus acidophilus, Lactiplantibacillus plantarum, Lactobacillus gasseri, and Lacticaseibacillus rhamnosus) was also assessed. Total polyphenols varied between 110.46 μg/g of fresh product (rhododendron honey) and 552.29 μg/g of fresh product (strawberry tree honey). Such result did not correspond to a parallel inhibitory α-glycosidase activity that, in each case was never higher than 33 μg/mL. Honeys were differently capable to fight the biofilm formation of the pathogens (inhibition up to 93.27%); they inhibited the in vitro adhesive process (inhibition up to 84.27%), and acted on mature biofilm (with values up to 76.64%). Their effect on bacterial metabolism was different too. Honeys were ineffective to inhibit E. coli mature biofilm nor to act on its metabolism. The action of the honey on probiotic strains seemed almost always stimulate their growth. Thus, these monofloral honeys might exhibit effects on human health and act positively as prebiotics.