Polyphenol oxidase (PPO) is the key enzyme responsible for enzymatic browning. To extend the shelf life of shredded lettuce, knowledge about biochemical PPO properties is required. The characterization of the enzyme from shredded, cold-stored lettuce was performed using pyrocatechol and the endogenous substrate (ES) (lettuce phenolics). The optimum pH and temperature for PPO activity were 5 and 50 °C, respectively. Natural infusions used as the PPO inhibitors (IC50) were ranked as follows: lovage (0.09%), marjoram (0.13%), orange peel (0.14%), oregano (0.15%), basil (0.22%), lemon peel (0.24%), parsley leaves (0.58%), and wheat bran (1.06%). Among well-recognized PPO inhibitors, kojic acid (0.00043%), ascorbic acid (0.00053%), and L-cysteine (0.00085%) were the most effective. Among the metal ions, MgCl2, FeCl2, and CaCl2 at 0.5 mM inhibited the PPO activity most effectively (by 28%, 27%, and 21%, respectively). The substrate used (pyrocatechol/ES) significantly influenced the enzyme inhibition. Using pyrocatechol, the lovage extract acted in a mixed mode (Kmi = 27.8 mM, Vmaxi = 2.03 mU), while the ES acted according to the non-competitive mode (Kmi= 0.57 mg GAE/mL, Vmax = 0.0046 U). The study confirms that natural extracts are more effective than L-cysteine when the ES is used. A pre-storage treatment with an infusion may be potentially used to improve the quality of shredded lettuce.