Enzymatic browning of sprouts during storage is a serious problem negatively influencing their consumer quality. Identifying and understanding the mechanism of inhibition of polyphenol oxidases (PPOs) in lentil sprouts may offer inexpensive alternatives to prevent browning. This study focused on the biochemical characteristics of PPOs from stored lentil sprouts, providing data that may be directly implemented in improving the consumer quality of sprouts. The purification resulted in approximately 25-fold enrichment of two PPO isoenzymes (PPO I and PPO II). The optimum pH for total PPOs, as well as for PPO I and PPO II isoenzymes, was 4.5–5.5, 4.5–5.0, and 5.5, respectively. The optimal temperature for PPOs was 35 °C. Total PPOs and the PPO I and PPO II isoenzymes had the greatest affinity for catechol (Km = 1.32, 1.76, and 0.94 mM, respectively). Ascorbic acid was the most effective in the inhibition of dark color formation by total PPOs, and showed ca. 62%, 43%, and 24% inhibition at 20-, 2-, and 0.2-mM concentrations. Ascorbic acid, l-cysteine, and sodium metabisulfite (20 mM) significantly inhibited color development in the reactions catalyzed by both isoenzymes of PPO. Ba2+, Fe3+, and Mn2+ (10 mM) completely inhibited PPO activity. This study of the effect of antibrowning compounds and cations on PPO activity provides data that can be used to protect lentil sprouts against enzymatic browning during storage and processing.