Pumpkin seed protein concentrate, a promising raw material with high biological value, holds potential for incorporation into bread technology. Protein structural changes occur during the bread-making process. This study aimed to investigate how pumpkin seed protein concentrate incorporated at different concentrations (5, 10, and 20%) affected the structural, mechanical properties, and conformational changes in protein substances within dough and bread made from wheat flour, utilizing near-infrared reflection spectroscopy. The gas- and shape-holding capacities of dough enriched and unenriched by pumpkin seed protein concentrate resulted similar. This aspect is important for the impact of the final product as bread on the consumers. The near-infrared reflection spectroscopy analysis revealed that the enrichment of bread with pumpkin seed protein concentrate not only introduced higher protein and unsaturated fat content in bread but also reduced the level of lignin, enhancing the digestibility of the final product. A deeper investigation into the nutraceutical and nutritional value of this enriched bread as well as of physio-chemical features investigated in the present work could be useful for the introduction of a new functional food in the market. Moreover, the enriched bread was acceptable in terms of sensory quality analysis, especially using 10% pumpkin seed protein concentrate. Therefore, to better understand the market potentiality of the enriched bread proposed in this study, future research should investigate how consumer acceptability could increase with a higher concentration of nutritional components perceived as beneficial for a lifestyle by specific consumer segments.