The purpose of this study was to investigate the antibacterial effect of 71 organic Sicilian honeys of different botanical origins against Staphylococcus aureus [American Type Culture Collection (ATCC) 9144™] and Pseudomonas aeruginosa (ATCC 27853™). The antimicrobial activity was determined by means of an agar diffusion assay from the estimation of the diameter of the inhibition zone produced by the honeys. S. aureus was more inhibited than P. aeruginosa (chi-square value 11.2698, P value 0.000788). In particular, a statistically significant inhibition growth against S. aureus was exhibited by the polyfloral (chi-square value 6.1714, P value 0.012983) and the eucalyptus honeys (chi-square value 4, P value 0.0455). Results partially suggest Sicilian organic polyfloral and eucalyptus honeys as possible alternatives to antimicrobial agents when S. aureus resistance causes failure of initial conventional antibiotic treatment.