Tofu has a high nutritional value, but it may also contain components that may have an antinutritional effect, such as trypsin inhibitors (TI), lectins and ureases. The aim of this study was to investigate the influence of the hydrothermal-cooking process of soybean in combination with commercial chymosin-pepsin rennet on the content and activity of TI, urease and lectins in tofu. High total TI content was found in tofu (5.00-16.87%). In addition, Kunitz (KTI = 3.52-4.32%) and Bowman-Birk (BBI = 5.00-12.53%) TI were registered, and BBI was detected in polymeric (1.38-2.71%) and monomeric (3.42-9.82%) forms. TI activity of tofu was very low (5.86-9.34%), corresponding to the very low activity of urease (0.51-3.07%). The percentage of lectin (2.62-4.63%) and urease (0.03-0.12%) in tofu was low. The results showed that the applied tofu production process is very effective in reducing the content and activity of TI, urease and lectin and provides the values without nutritional harmful effect.