Honey is a natural sweetener made by bees that exhibits antimicrobial activity, mainly related to its H2O2 content. The aim of this work was to research the H2O2 concentration of 24 Spanish honeys from different botanical origins, studying their possible correlation with glucose oxidase (GOx), catalase (CAT), and anti-Staphylococcus aureus activities (minimal inhibition concentration (MIC), minimal bactericidal concentration (MBC), and percentage of inhibition at 5% (w/v) honey against Staphylococcus aureus), as well as possible correlations among all the analyzed parameters. The results showed that the H2O2 concentration did not depend on the botanical origin of the honeys. There were neither correlations between the H2O2 concentration and the activities of GOx and CAT, nor between GOx and antimicrobial activity. However, CAT and antimicrobial activities were positively correlated. Therefore, CAT could be successfully used as a possible marker of the antimicrobial activity of honeys against Staphylococcus aureus. Furthermore, a linear regression model has been fitted to explain the antimicrobial activity from CAT and GOx activity and H2O2 concentration. Although H2O2 is one of the compounds involved in honey’s antibacterial activity, this capacity also strongly depends on other honey components (such as low water activity, acidity, osmolarity, and phenolic compounds). The very high anti-Staphylococcus aureus activity exhibited by all samples could be interesting for commercial honey-based formulations also helping to promote local beekeeping.