Background: Honey is a natural biologically active substance with antibacterial, antioxidant, anti-inflammatory, and tissue regeneration–promoting properties, and it has traditionally been used in wound treatment for centuries. Numerous studies have confirmed that the phenolic compounds, enzymes, hydrogen peroxide, and amino acids contained in honey inhibit bacterial growth and accelerate wound healing. Wound infections are commonly caused by pathogenic bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli which are major factors that delay wound healing and lead to complications. However, the direct application of honey to wounds presents challenges such as adhesiveness, absorption, dose stability, and sterility. Therefore, it is necessary to formulate honey into a suitable pharmaceutical dosage form to facilitate its use and introduce natural raw materials into therapeutic practice. Thus, incorporating honey as the main active ingredient into a hydrogel dressing could enable the development of a novel therapeutic wound dressing that is physically and chemically stable and capable of maintaining an optimal moist wound environment, which is essential for wound healing. Honey-containing hydrogel dressings differ from other dressings in their ability to balance the wound environment, absorb and retain moisture, provide antibacterial and anti-inflammatory effects, and support tissue regeneration. The significance of this study lies not only in investigating the antibacterial activity of honey but also in determining the bacterial killing time, thereby providing useful data for future studies. Aim: To investigate the antibacterial activity of honey and determine its bacterial killing time. Materials and Methods: Domestic honey samples were compared with imported honey samples in terms of antibacterial activity and bacterial killing time. Domestic honey samples were coded as D1, D2, D3, C1, and C2, while the imported honey sample was coded as M. A total of six honey samples were evaluated. Using the broth microdilution method, the antibacterial activity against four bacterial strains commonly associated with burn wound infections Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 28583, Klebsiella pneumoniae ATCC 1706, and Escherichia coli ATCC 25218 was determined and compared. Subsequently, bacterial killing time was evaluated using the broth dilution method with honey concentrations prepared at ratios of 7:3 (70%), 5:5 (50%), and 4:6 (40%). Results: The antibacterial activities of D1, D2, D3, C1, C2, and M honey samples against S. aureus, P. aeruginosa, K. pneumoniae, and E. coli were determined. Among them, D3, C2, and M honey samples showed stronger antibacterial activity. Therefore, these three honey samples were further investigated by determining bacterial killing time over an 18-hour period. For D3 honey diluted at a 7:3 ratio, the killing time was 15 hours for S. aureus and 7 hours for P. aeruginosa, while the killing times for K. pneumoniae and E. coli exceeded 18 hours. D3 honey diluted at a 5:5 ratio showed bacterial killing times exceeding 18 hours for all tested bacteria. For C2 honey diluted at a 7:3 ratio, the killing times were 16 hours for S. aureus, 12 hours for P. aeruginosa, 16 hours for K. pneumoniae, and 17 hours for E. coli. C2 honey diluted at a 5:5 ratio killed E. coli in 18 hours, whereas the killing times for S. aureus, P. aeruginosa, and K. pneumoniae exceeded 18 hours. For M honey diluted at a 7:3 ratio, the killing times were 8 hours for S. aureus, 7 hours for P. aeruginosa, 9 hours for K. pneumoniae, and 8 hours for E. coli. M honey diluted at a 5:5 ratio killed S. aureus in 9 hours, P. aeruginosa in 10 hours, K. pneumoniae in 10 hours, and E. coli in 9 hours. Conclusions 1. The antibacterial activity of honey against Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli varied depending on honey concentration, with honey concentrations of 40–45% demonstrating the highest antibacterial activity. Furthermore, M honey exhibited the strongest antibacterial activity compared to the other honey samples. 2. In determining bacterial killing time, the domestic honey samples killed bacteria within 9 hours, whereas the imported honey sample killed bacteria within 7 hours.