In the past decades, honey has been subjected to various laboratory and clinical investigations. The antimicrobial properties of honey have been attributed to both the hydrogen peroxide as well as non-peroxide components. Although the antimicrobial activities of Malaysian tualang honey have been established against an extensive spectrum of microorganisms, no studies have investigated honey components that essentially contributed to these activities. The objectives of this study were to fractionate tualang honey into polar, acidic and basic fractions, using solid phase extractions (SPE) and to determine the antimicrobial activity of the various fractions against common and multidrug resistant wound and enteric bacteria. Our study demonstrated that the acidic fractions of honey gave lower MIC values and therefore contributed to a large proportion of its antimicrobial properties. This study also demonstrated that fractionated honey gave remarkable bactericidal activities for Stenotrophomonas maltophilia, Streptococcus agalactiae, Escherichia coli and Acinetobacter baumannii when compared to the non-fractionated honey. However, fractionating honey into different portions does not enhance tualang honey's antibacterial activities against five bacterial species (coagulase-negative staphylococci, Methicillin-resistant Staphylococcus aureus (MRSA), Stenotrophomonas maltophilia, Acinetobacter baumannii and Enterobacter cloacae). Since our results demonstrated that the bactericidal activities of tualang honey were largely enhanced in the acidic fractions of the honey, future work should focus on the acidic fractions of tualang honey to explore the main constituents that contribute to this effects.