Background: Honey possesses therapeutic properties that are the result of a range of factors including high sugar content, low pH, hydrogen peroxide, and bee-derived peptides. Honey also contains antimicrobial phytochemicals, which represent a rich source of leads for the development of drugs for the treatment of microbial infections. Honey samples donated by UK beekeepers (n = 217) and Manuka samples (n = 3) were screened for the presence of novel antibacterial compounds by determining activity against methicillin-resistant Staphylococcus aureus (MRSA). The majority (88%) of the honeys showed inhibitory activity; two of these samples were found to contain potentially novel antibacterial compounds, which were likely to be plant-derived. The pollen present in honey represents a record of the plants that contributed to the making of the honey and may be the source of specific antibacterial factors. For this reason, pollen was extracted and characterised from honey samples using microscopic analysis and rbcL DNA metabarcoding (454 and Illumina). Results: DNA metabarcoding provided superior discrimination for some plant families and greater repeatability compared to microscopic analysis. Key species identified in the antibacterial samples included woodruff (Galium odoratum), bluebell (Hyacinthoides non-scripta), and dandelion (Taraxacum officinale). Extracts from active honeys and characterised plants demonstrated antibacterial activity against MRSA, E. coli, and P. aeruginosa. Activity-guided characterisation using a thin layer chromatography/mass spectrometry interface highlighted the presence of some known and some novel compounds. Significance: Understanding the floral composition of honey has a wide variety of applications. DNA metabarcoding pollen extracted from honey allows us to identify the plant species that may be contributing to the antimicrobial effect of honey. The demonstration of antibacterial activity may provide new lead compounds that could serve as selective agents against MRSA and other antibiotic-resistant bacteria.