Geranium wallichianum is a medicinal plant belonging to the Geraniaceae family. It is mainly found in the East Asian Himalayas. Because of its numerous ethnomedicinal uses in the community, this plant is threatened by over-exploitation and over-harvesting. Pakistan has already lost its population by more than 75%. Considering its medicinal importance, this plant must be conserved before it becomes extinct. This study aimed to provide the protocol for in vitro conservation of G. wallichianum. Owing to the plant conservation applications of osmotic stress inducers such as polyethylene glycol (PEG) and sucrose, their ability to regulate the in vitro growth of this medicinally significant plant was evaluated. Using nodal segments as explants, the effect of Murashige and Skoog (MS) medium supplemented with different concentrations of PEG (0, 0.5, and 1.5%) and sucrose (0, 4, and 8%) on various growth parameters was investigated. At 45-day intervals, PEG (0.5 and 1.5%) treatment was found to be more effective than sucrose treatment in reducing in vitro growth (0.92 and 0.57 cm), with minimum fresh (0.06 and 0.04 g) and dry weight (0.04 and 0.02 g), respectively. Total soluble sugars (4.07 and 4.12 mg/g) and total phenolic content (0.96 and 0.19 mg/g) were significantly increased by PEG treatment (0.5 and 1.5%, respectively), whereas total chlorophyll content (0.17 and 0.14 mg/g) and total protein content (2.38 and 2.32 mg/g) were decreased. In addition, Pearson correlation analysis confirmed that total phenolic content was negatively correlated with chlorophyll a, chlorophyll b, and total chlorophyll content, whereas shoot length was negatively correlated with total phenolic content but positively correlated with fresh weight and dry weight. According to the principal component analysis, total phenolics and total sugars, which clustered distinctly from the other parameters, indicated relatively higher osmotic stress responses to PEG and sucrose. Based on these findings, it appears that PEG was more effective than sucrose at slowing in vitro growth during short-term conservation.