Background: As offices change and employees spend more of their lives at work, managers and designers need to understand the relationship between employees and their work environment to spend investment dollars wisely. This study investigated the efficacy of various physical workstation environment characteristics in a medical office building (MOB) using observation and employee survey responses. Method: This was a cross-sectional exploratory study measuring both the observed physical work environment and employees’ perceptions of their workstation satisfaction, comfort, and performance in a MOB. Results: Findings suggest that employees seek to improve comfort ratings for acoustics, privacy levels, and access to daylight and nature views. No moderating nor main effects were seen for satisfaction or performance, but comfort ratings did show main three main effects. Discussion: The study highlighted opportunities to enhance employee comfort, performance and satisfaction using the physical environment. These findings inform and identify future research opportunities for MOB design.