The antibacterial activity of 31 Greek and Cypriot honeys against Staphylococcus aureus and Pseudomonas aeruginosa was initially screened using an agar-well diffusion assay in comparison with manuka honey. The minimum inhibitory concentration (MIC) was determined in broth using a spectrophotometric-based assay. The MIC of treated honeys with catalase or proteinase K was determined and compared with those of untreated honeys. All tested honeys demonstrated antibacterial activity against S. aureus on agar-well diffusion assay. MICs of tested honeys were determined as 3.125-25% (v/v), compared with manuka honey at 6.25% (v/v). Similarly, 21 of 31 tested honeys demonstrated antibacterial activity on agar-well diffusion assay against P. aeruginosa. Their MICs ranged from 6.25% to 25% (v/v) compared with 12.5% (v/v) for manuka honey. Antibacterial activity of tested honeys could be largely attributed to hydrogen peroxide formation and in some cases to unidentified proteinaceous compounds. In conclusion, Greek and Cypriot honeys demonstrated significant but variable antibacterial activity against P. aeruginosa and especially S. aureus. To the best of our knowledge this is the first study that has thoroughly examined the antibacterial activity of Greek and Cypriot honeys compared with manuka honey. The high antibacterial activity exerted by some tested honeys warrants further investigation.