Hyperhydricity syndrome (HHS) occurs when plants are stressed resulting in sharp changes in the ethylene concentration in the in vitro culture. One of the characteristics of HHS is the reduction of cell wall lignification. The aim of this study was to examine the effects of silver nanoparticles (AgNPs) on HHS in micropropagated Thymus daenensis shoots. AgNPs (0, 1, 2.5, or 5 mg L −1 ) were mixed with Murashige and Skoog (MS) culture medium and the antioxidant response and variations in lignin content were measured on micropropagated shoots derived from seedlings cultured with AgNPs. In addition, the different forms of microshoot polyamines (PAs) were investigated through High Performance Thin Layer Chromatography (HPTLC). The results indicated that seedling pretreatment with AgNPs at concentrations of 1 and 2.5 mg L −1 led to a decrease in the activity of the microshoot antioxidant system resulting in an increase of H 2 O 2 . Furthermore, the increased lignin content in response to the 2.5 mg L −1 AgNPs treatment was accompanied by an increase in the concentration of the bonded form of putrescine (Put) and spermine (Spm). There was a direct correlation between the lignin content and the concentration of bonded PAs. We hypothesize that the higher level of bonded PAs reduced the concentration of the free forms, leading to increased lignification. This may have led to the decreased HHS observed in shoot cultures.