Background This study compared the chemical, morphological and microhardness changes in carious dentine following application of NaOCl‐based (Carisolv), papain‐based (Papacarie) chemomechanical caries removal agents with conventional rotary method. Methods Thirty‐two carious and eight non‐carious human permanent molars were used. The carious molars were randomly distributed into four groups: Group 1 (positive control) – molars were left without caries removal; Group 2 – caries excavated with rotary method; Group 3 – caries excavated with Carisolv; Group 4 – caries excavated with Papacarie. Group 5 (negative control) consisted of eight sound molars. After caries excavation, all specimens were prepared for chemical, morphological analysis and Vickers hardness testing. Results EDX analysis showed no significant difference in Ca wt%, P wt% and Ca/P ratio among Groups 2 to 5. Vickers hardness of dentine from Groups 3 and 4 was significantly lower (p < 0.05) than for Groups 2 and 5. The use of Papacarie resulted in a dentine surface without smear layer and patent tubules, while Carisolv resulted in a dentine surface exhibiting two patterns: (1) the presence of smear layer or (2) patent tubules with partial smear layer formation. Conclusions Papain‐based (Papacarie) chemomechanical caries removal method is a reliable alternative to conventional rotary and NaOCl‐based (Carisolv) caries removal.