This study evaluated the antimicrobial activity of different levels of HMF value of honey at different concentration (15,20and 25%honey). Honey and HMF concentrations of 15 %, 20 % and 25 % honey in distilled water were prepared. These preparations were tested for antimicrobial activity against four different types of pathogenic bacteria; Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Bacillus subtilis . Antimicrobial activity was determined as an equivalent of the inhibition zones diameters using filter paper discs (in millimeters) after incubation of the cultures at 30°C for 24 hours for bacterial species. The results showed a bactericidal activity of HMF value of honey against a broad spectrum of bacteria with variable degrees. By use of least significant differences test for multiple comparisons, it is apparent that the honey of HMF value (90.24 mglkg) showed the highest inhibitory effect on bacterial growth compared to other honey treatments and HMF concentrations. This value (90.24 mglkg) had the highest bactericidal activity against Escherichia coli at concentrations of 15and 20% honey. Similarly, the HMF (90.24 mglkg) value had significantly higher inhibitory effects compared to other treatments on Bacillus subtilis growth at concentration 15, 20 and 25% honey and on Staphylococcus aureus growth at concentration 15and 25% honey. However, there was no inhibitory effect of HMF value of honey (203.0 mglkg) against all bacterial strains tested at concentration 15% honey. Considerably, no inhibitory effect of all HMF values of honey treatments was noted on Pseudomonas aeruginosa. Conclusion, the different levels of HMF value of honey at different concentration (15,20and 25%) showed a bactericidal activity against a broad spectrum of bacteria with variable degrees