The aim of the study was to evaluate the effects of the antioxidant N-acetylcysteine (NAC), added in freezing/thawing solutions, on reactive oxygen species (RRS) levels and on ovarian tissue preservation after cryopreservation. Ovarian samples from 10 subjects suffering from cancer diseases were cryopreserved using the slow freezing/rapid thawing standard protocol without or with NAC supplementation. RRS levels produced during cryopreservation were monitored by electron paramagnetic resonance (EPR) spectroscopy. The preservation of fresh ovarian tissue (t0), thawed tissue (t1 and t1 NAC) and thawed tissue maintained at 4°C for 2 hrs (t2 and t2 NAC) was analysed by light microscopy, transmission electron microscopy, Ki67 immunohistochemical and TUNEL analysis. It was possible to design a maximum peak for RRS production at t1, which slightly decreased at t2. NAC reduced the extent of RRS levels in cryopreserved ovarian tissues if compared with non-supplemented ones, although not restoring RRS production to baseline values. Comparative analysis between the two cryopreservation protocols showed that a better preservation of morphological characteristics, proliferation index and DNA integrity of ovarian tissue was obtained using NAC and no differences between t1NAC and t2NAC were observed. The employment of NAC during cryopreservation procedure could be an useful strategy for preserving the function of endogenous cellular systems. Nevertheless, further studies on the viability of thawed ovarian tissue are needed to support the feasibility of this approach in clinical settings.