| Summary: | This study aimed to investigate the effects of different environmental factors (temperature, pH, and NaCl) and food matrices (skimmed milk powder, lecithin, and sucrose) on the antibacterial activity of theaflavins (TFs) against <i>Staphylococcus aureus</i> (<i>S. aureus</i>) and <i>Salmonella paratyphi B</i> (<i>S. paratyphi B</i>). TFs showed a larger diameter of inhibition zone (DIZ, 12.58 ± 0.09 mm–16.36 ± 0.12 mm) value against <i>S. aureus</i> than that of <i>S. paratyphi B</i> (12.42 ± 0.43 mm–15.81 ± 0.24 mm) at the same concentration (2–10 mg/mL). When temperatures were 25–121 °C, the DIZ of TFs against both <i>S. aureus</i> and <i>S. paratyphi B</i> was not significantly different. As pH increased from 2 to 10, their DIZ values decreased significantly from 16.78 ± 0.23 mm to 13.43 ± 0.08 mm and 15.63 ± 0.42 mm to 12.18 ± 0.14 mm, respectively. Their DIZ values increased slightly as the NaCl concentration increased from 0.2 mol/L to 0.8 mol/L, while their DIZ values decreased significantly for skimmed milk powder concentrations in the range of 20–120 g/L. Regarding the concentrations of lecithin and sucrose were 2–12 g/L and 10–60 g/L, their DIZ values showed no significant change against <i>S. paratyphi B</i>, but an increased trend for <i>S. aureus</i>. Under the above different environmental factors and food matrices, TFs maintained excellent antibacterial activity against <i>S. aureus</i> and <i>S. paratyphi B</i>, providing a theoretical guidance for applying TFs as novel antibacterial additives in the food industry.
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