In this work, the nitrided layer on austenitic AISI 316L stainless steels were examined by means of in situ electrochemical nanoindentation, and the effect of electrochemically charged hydrogen on the mechanical properties were investigated. By using this method, changes in the mechanical properties due to the absorption of atomic hydrogen at different nitride layer depths have been traced. One important finding is that hydrogen charging of the nitriding layer can soften the layer and reduce the layer hardness, and the reduction of the nitrogen concentration in the nitride layer reduces this softening effect.