When AISI 316L stainless steels are submitted to the nitriding process at temperatures lower than 450 °C, a high nitrogen content expanded austenite phase is formed, which shows higher hardness and higher pitting corrosion resistance compared to the untreated material. As a result, this material becomes adequate for biomedical application. The conditions of the nitriding technique, such as gas mixture, pressure, time and temperature, play an important role in some properties of the modified layer, including: thickness, hardness and N concentration along the layer. This paper explores a set of six samples of AISI 316L, nitrided at different times and temperatures, whose properties show important differences. The aim of this research is to investigate the correlation between the nitrided layer thickness (in the range of 0.77 to 11 µm) with both X-ray patterns characteristics and hardness measurements, which used two distinct loads. The results of this study show that: whereas the 3.6 gf load was suitable to measure the real hardness for four of the nitrided layers showing thickness ≥ 2.9 µm, the 50 gf load measured a substrate contribution, probably even for the highest thickness, 11 µm. Moreover, analyzing different reflections of the X-ray patterns showed evidence of the clear consistency between the X-Ray depths and the nitrided layer thicknesses: if the layer thickness is lower than the penetration depth of X-rays, two phases (austenite and expanded substrate) are present. If the layer thickness is higher, only the austenite is observed. Finally, concerning the citotoxicity property, all the samples, nitrided or not, were approved in the test for biocompatibility, indicating their potential use for biomedical applications.