The aim of the present study was to evaluate adolescents’ acceptability of a novel flat bread modified by substituting a part of the wheat flour content with a Pleurotus ostreatus powder rich in β-glucans, which can potentially provide health benefits. The effects of food technology neophobia and adolescents’ food habits on hedonic perception of the developed product was also investigated. Two hundred and two adolescents (age range: 13–18 years; girls: 49.5%; boys: 50.5%) evaluated their liking of two flat breads, one with mushroom powder added and one control sample with only wheat flour. Sample acceptance was studied in relation to age, gender, neophobic traits and healthy food habits. The results showed that, even if the sample with mushroom powder added was generally well accepted, there were different hedonic responses among adolescents according to their food technology neophobia level and healthy habits. In particular, adolescents with a low food technology neophobia level and healthy eating behavior mostly appreciated the sample with mushroom powder added, whereas subjects with neophobic and unhealthy eating behavior gave comparable hedonic scores to the two samples. Moreover, a negative correlation was found between food technology neophobia level and healthy food habits. In conclusion, it is possible to develop a β-glucan-enriched product appreciated by adolescents using a sustainable ingredient. The developed product may be used to achieve the daily recommended intake of β-glucans by adolescents.