Mass timber construction has recently gained popularity due to its outstanding environmental benefits and building performance, which hold revolutionary potential for the construction industry. However, its impacts from the perspective of occupants have not been thoroughly explored. This study introduces an innovative empirical approach that explores the potential benefits of mass timber construction for individuals and organizations, with an emphasis on the workplace. We review the conceptual framework regarding how visual and physical exposure to timber construction materials and finishes have a positive effect on individuals and organizations at a broad level. We propose a more holistic mixed-method behavioral approach to studying occupant behavior and well-being by integrating self-reported questionnaires, objective biomarkers (heart rate variability and hair cortisol), and indoor environmental quality (IEQ) measures. Our study offers a novel research primer on the exploration of mass timber construction impacts and benefits for both office workers and construction workers. Participants from different office settings completed pre- and post-occupancy evaluation surveys to assess their experiences, including IEQ satisfaction, productivity, and health. Office workers were located in three different offices: a controlled laboratory environment, an open-plan office, and an open-plan space with a timber interior. The construction workers worked in a timber space for three months and then moved to work in a building with a concrete structure. The analysis included descriptive statistics, t-tests, ANOVA, and linear regression to compare differences between office settings and assess the relationship between environmental variables and overall satisfaction in IEQ, comfort, productivity, and health. In office workers, in terms of building image, thermal comfort, and artificial lighting, the data analysis revealed significant differences in occupants’ satisfaction levels between office settings. However, the low number of participants affected the results, and some factors were not found significant in relation to the office setting. Among tradespeople, there was no relationship between the building environment and productivity, health, or comfort. However, the results of hair cortisol testing indicated that working in a timber space can decrease the level of cortisol (stress) and have an impact on the productivity of workers. Such occupant’s perspective research is pivotal to informing policy makers, developers, business owners, construction professionals, timber industry stakeholders, environmentalists, and researchers in their decision-making processes. Fostering the future widespread adoption and advancement of mass timber construction.