Strawberry is a susceptible plant to water stress, and its growth is severely declined under water shortage. Therefore, the aim of this study was to assess the impacts of iron, as a micro-nutrient, in the form of a nano-oxide on the tolerance of strawberry plants to water stress conditions under in vitro culture. Three levels of PEG-induced water stress (0, 5%, and 7% w v(-1) PEG 6000) and three levels of iron nano-particles (0, 1, and 1.2 mg L-1) were supplemented to Murashige and Skoog (MS) culture medium. The results demonstrated that PEG-induced water stress negatively affected shoot fresh and dry weight, root fresh and dry weight, and physiological traits such as relative water content, membrane stability index, and pigment content. The biochemical traits were also affected by PEG-induced water stress; consequently, antioxidant enzyme activity, leaf proline content, total soluble sugar, and malondialdehyde (MDA) and H2O2 content were increased. The application of iron nano-particles exhibited the capability of compensating for the negative impacts of PEG-induced water stress on the morphological and physiological traits, increased antioxidant enzyme activity, and decreased MDA and H2O2. Principal component analysis (PCA) indicated that the relationships among all the measured traits were influenced by nano-iron application. These relationships signified the importance of nano-iron application regarding strawberry plants under in vitro and PEG-induced water stress conditions. According to the morphological traits, a medium level of iron nano-particles (approximately 1 mg L-1) is well capable of improving the tolerance of strawberry plants under PEG-induced water stress and in vitro conditions.