Button mushrooms (Agaricus bisporus), one of the most common edible mushroom species, are sensitive to damages because of the absence of a protective skin layer and have a limited shelf life. Osmotic dehydration (OD), mainly used as a pre-processing step of conventional preservation methods, has been proposed as an efficient, mild treatment to preserve mushroom superior quality. In this study, response surface methodology, coupled with a Box–Behnken design, was used to investigate the effect of glycerol concentration (30–50%), temperature (30–50 ℃), and duration of osmosis (0–180 min) in order to optimize the process prior to a subsequent freezing step. For each response, including mass transfer and selected quality indices, a second-order polynomial model was developed, and all process factors were found to have a significant impact. Based on the desirability approach and pre-set criteria, optimum operating conditions were estimated, namely osmosis at 50 °C, for 120 min, with a 42% glycerol solution, and the corresponding validation experiments were performed. Based on the error estimated between experimental and predicted values, polynomial equations were found to adequately predict parameter values. Based on a shelf-life test under frozen storage, OD-treated samples retained better quality attributes compared to their untreated counterparts.