Oyster mushroom (Pleurotus ostreatus) is the second most widely cultivated mushroom worldwide but has a short shelf-life due to its high moisture content. Osmodehydrofreezing, including a step of osmotic dehydration (OD) prior to freezing, can be applied to extend product shelf life and preserve its quality characteristics. In this study, the effect of glycerol concentration (30–50%), temperature (30–50 °C), and immersion time (20–190 min) on mass transfer properties (WL, SG, aw, %moisture content, salt intake) and important quality indices (color and texture) was investigated. Experiments were conducted using a Box–Behnken Design with three factors at three levels, second order equations were used to describe all measured parameters (following the Response Surface Methodology principles), and preselected desirability functions were used to set the acceptability criteria for optimization. The optimized conditions were found to be 38.7 °C, 30% glycerol, and 130 min of immersion time. To maximize OD effect, an initial step was investigated, using the side streams of rose flowers distillation (ODR samples), aiming at enriching samples with bioactive compounds. Frozen OD and ODR samples demonstrated a significant improvement in color and texture retention, and suffered from a reduced drip loss after thawing, compared to their untreated counterparts.