The concept of conserving healthy tooth structures during cavity preparation has gained popularity with chemo-mechanical caries removal. This study compared three methods of caries removal using: a chemo-mechanical caries removal papain gel; Papacarie® (these contain natural ingredients, mainly papain enzyme); and mechanical preparation with a bur rotary instrument. The purpose of this study was to compare affected dentin micro-hardness after removal of infected dentin with mechanical and chemo-mechanical techniques. Twenty-seven permanent molar teeth were randomly divided into three groups receiving removal of infected dentin. These were: Group 1: chemo-mechanical technique using papain gel; Group 2: chemo-mechanical technique using Papacarie®; Group 3: mechanical technique using a bur rotary instrument. Each group was tested using Knoop Micro-hardness tester, and the data were submitted to one way ANOVA and Post-hoc Tukey test. There is a significant difference between Groups 1 and 3, and Groups 2 and 3, p = 0.000. However, there is no significant difference between Groups 1 and 2, p = 1.000. Affected dentin micro-hardness after removal of infected dentin with a bur rotary tool is higher than after use of the papain gel or Papacarie®. Affected dentin micro-hardness after removal of infected dentin with Papacarie® and papain gel give almost the same result.