Antimicrobial resistance, driven by antimicrobial misuse and declining therapeutic innovation, poses a critical global health challenge. This study evaluated the physicochemical profiles and antimicrobial efficacy of five monofloral Pakistani honeys, namely Helianthus annuus L. (Sunflower), Ziziphus jujuba (Beri), Acacia modesta (Phulai), Justicia adhatoda L. (Baikar), and Brassica spp. (Mustard), alongside Leptospermum scoparium (Manuka) honey containing methylglyoxal (MGO 550+), against Staphylococcus aureus American Type Culture Collection (ATCC 6538) and Escherichia coli (ATCC 8739). Physicochemical properties were assessed according to AOAC guidelines, whereas antibacterial activity was determined using agar well diffusion and broth microdilution assays. Quality parameters across the honey varieties met international standards, with ranges of 28.9-78.2 mm Pfund for color, 3.36-4.20 for pH, 29.6-43.5 meq/kg for acidity, 16.0%-19.2% for moisture, 0.14-0.43 g/100 g for ash, 6.0-6.7 g/100 g for sucrose, 76.0-81.6 g/100 g for total sugars, 0.28-0.65 mS/cm for electrical conductivity, and 21.2-40.0 mg/kg for hydroxymethylfurfural. Proline content (≥180 mg/kg), diastase numbers (DN) (10.0 ± 0.46 to 39.32 ± 0.58 DN), and invertase activity IN (63.00 ± 8.70 to 75.33 ± 5.70 IN) confirmed the freshness and purity of the honeys. In diffusion assays, Manuka honey produced the largest inhibition zones against E. coli (13.40 mm) and S. aureus (14.40 mm). Among the local varieties, Mustard honey showed strong activity against E. coli (13.40 mm), whereas Baikar honey showed the greatest activity against S. aureus (13.90 mm); Sunflower honey showed the lowest efficacy against E. coli (8.90 mm). The lowest minimum inhibitory concentrations (MICs) were recorded for Manuka honey (7.8% w/v against S. aureus and 16% w/v against E. coli), whereas Phulai and Mustard honeys required concentrations of up to 17.5% w/v. These findings support the traditional use of Pakistani honeys as potential complementary antibacterial agents and highlight the need for comprehensive in vivo and clinical evaluations.