BACKGROUND: Rosa damascena is extensively cultivated in various regions of Iran due to its aesthetic attributes, medicinal qualities, and essential oil production. This study investigated the efficacy of Ascophyllum nodosum extract (AnE) at concentrations of 0, 2, and 3 g L- 1 and Nano-silicon (nSiO2) at concentrations of 0, 50, and 100 mg L- 1 in ameliorating the impact of salinity on two genotypes of Damask rose ('Chaharfasl' and 'Kashan') under in vitro culture conditions. Additionally, various physio-chemical characteristics of R. damascena explants were assessed. RESULTS: The findings revealed that exposure to 100 mM NaCl resulted in a substantial reduction in the Relative Water Content (RWC), Membrane Stability Index (MSI), leaf pigments (Chlorophyll b, Chlorophyll a, total Chlorophyll, and carotenoids), chlorophyll fluorescence parameters, and protein content in both genotypes when compared to control conditions. Salinity induced a significant increase in the parameter F0 and a decrease in the parameter Fv/Fm compared to the control conditions in both genotypes. Nonetheless, the genotype Kashan treated with 3 g L- 1 AnE + 100 mg L- 1 nSiO2 exhibited the maximum Fm value under control conditions, with a significant difference compared to other treatments. Furthermore, salinity caused a considerable reduction in Fm in both 'Kashan' and 'Chaharfasl' by 22% and 17%, respectively, when compared to the control condition. 'Kashan' displayed the maximum Fv/Fm compared to the other genotype. The maximum levels of Malondialdehyde (MAD) and hydrogen peroxide (H2O2) were also observed in explants affected by salinity. The combination of 3 g L- 1 AnE + 100 mg L- 1 nSiO2, followed by 2 g L- 1 AnE + 100 mg L- 1 nSiO2, exhibited substantial positive effects. Salinity also led to an increase in proline content and the activity of peroxidase (POD), superoxide dismutase (SOD), guaiacol peroxidase (GPX), and catalase (CAT) in both genotypes. The activity of these enzymes was further enhanced when AnE was applied at concentrations of 2 and 3 g L- 1 in combination with 100 mg L- 1 nSiO2. CONCLUSIONS: The 'Kashan' genotype displayed greater tolerance to salinity by enhancing water balance, maintaining membrane integrity, and augmenting the activity of antioxidant enzymes compared to 'Chaharfasl'. The utilization of nSiO2 and AnE biostimulants demonstrated potential benefits for R. damascena, both under salinity and control conditions. These findings hold substantial importance for researchers, policymakers, and farmers, offering valuable insights into the development of salinity-tolerant crop varieties.