Because of its powerful therapeutic potential in wound care, honey has been given much attention. One of the most relevant and researched characteristics of honey is its multifactorial antibacterial activity. Several antibacterial compounds have been identified including hydrogen peroxide (H2O2). The goal of the study was to characterize the antibacterial potential of honeys from various botanical origins (n = 233) against Staphylococcus aureus and Pseudomonas aeruginosa and establish the H2O2 content. Five types of honey were identified: acacia (n = 72), linden (n = 23), rapeseed (n = 9), sunflower (n = 8) and multi-floral (n = 121). The overall antibacterial activity of all the honey samples, expressed as a minimal inhibitory concentration of honey, strongly correlated with the level of H2O2. On the other hand, linden honey, which exhibited the strongest antibacterial effect, showed weak or no correlation between the antibacterial activity and the level of H2O2, depending on bacterial strain. The weakest antibacterial activity was documented in the rapeseed honey samples and it was associated with the low content of H2O2. These data suggest that antibacterial activity of honey is mainly H2O2-dependent; however, certain type of honey such as linden honey, showed a strong antibacterial effect which, may be, at least partially, attributed to non-peroxide factor(s).