Background: Honey is a versatile and complex substance consisting of bioactive chemicals which vary according to many bee and environmental factors. The aim of this study was to assess the physical and antimicrobial properties of five honey samples obtained from three species of bees; two stingless bees, Frieseomelitta nigra and Melipona favosa and one stinging bee, Apis mellifera (fresh and aged honey). Samples were acquired from apiaries across Trinidad and Tobago. An artificial honey, made from sugar, was also used for comparison. Methods: Physical properties such as appearance, pH, moisture content, sugar content and specific gravity were determined. Antimicrobial activity was assessed utilizing the agar diffusion assay and comparison to a phenol equivalence. The broth microdilution test was performed to determine the minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs) of the five honey samples against four common pathogens, including Staphylococcus aureus, Escherichia coli, Streptococcus pyogenes and Haemophilus influenzae. Results: All honey samples were acidic, with pH values ranging from 2.88 (M. favosa of Tobago) to 3.91 (fresh A. mellifera). Sugar content ranged from 66.0 to 81.6% with the highest values detected in stinging bee honeys of the A. mellifera (81.6 and 80.5°Bx). Moisture content ranged from 16.9% for aged A. mellifera honey (from Trinidad) to 32.4% for F. nigra honey (from Tobago). The MICs (2 to 16%) and MBCs (2 to 32%) of stingless bee honeys were lower than that of stinging bee and artificial honeys (16 to > 32%). Stingless bee honeys also exhibited a broad spectrum of antimicrobial activity against both Gram-positive and Gram-negative organisms with higher phenol equivalence values (4.5 to 28.6%) than the A. mellifera honeys (0 to 3.4%) against the isolates tested. M. favosa honey of Tobago displayed the greatest antimicrobial activity as indicated by the high phenol equivalence and low MIC and MBC values. Conclusions: Stingless bee honeys from Tobago showed the greatest antimicrobial activity when compared to the other honeys used in this study. M. favosa honey of Tobago showed the most potential for use as medicinal honey.