<p>Background: It is demonstrated that optimal preincubation time improves oocyte quality, fertilization potential and developmental rate. This study aimed to evaluate the effect of preincubation time in the simple and myo-inositol supplemented medium on the oocyte quality regarding oxidative stress and mitochondrial alteration.<br /> Methods: Cumulus oocyte complexes (COCs) retrieved from superovulated NMRI mice were divided in groups of 0, 4 and 8 <em>hr</em> preincubation time in the simple and 20 <em>mmol/L</em> myo-inositol supplemented media. Intracellular reactive oxygen species (H₂O₂), glutathione (GSH), mitochondrial membrane potential (MMP), ATP content, and mitochondrial amount were measured and analyzed in experimental groups. One-way ANOVA and Kruskal-Wallis were respectively used for parametric and nonparametric variables. Statistical significance was defined as p&lt;0.05.<br /> Results: In comparison to control group, variables including ROS, GSH, mitochondrial amount, fertilization and developmental rates were significantly changed after 4 <em>hr</em> of preincubation in the simple medium, while MMP decreased following 8 <em>hr</em> of preincubation in the simple medium (p˂0.001). Preincubation of oocytes up to 8 <em>hr</em> in the simple medium could not decrease ATP content. For both 4 and 8 <em>hr</em> preincubation times, myo-inositole could decrease H₂O₂ and increase GSH and MMP levels and consequently could improve fertilization rate compared to oocytes preincubated in the simple culture.<br /> Conclusion: It seems that 4 <em>hr</em> or more preincubation time can decrease the oocyte quality and lead to reduced oocyte fertilization and developmental potential. Howevere, myo-inositol may prevent oocyte quality reduction and improve fertilization potential in comparision to the equivalent simple groups.</p>