The objective of this study was to assess the internal quality of a university hospital by health professionals and its relation to job satisfaction. The survey was conducted in a university hospital in Attica. The sample was 131 employees consisting of medical, nursing, administrative and paramedical staff, with the method of random sampling. The collection of the data lasted from October to December 2016. The questionnaire used in this survey was the SERVQUAL tool with five dimensions: reliability, responsiveness, tangibles, assurance, and empathy. Additionally, five job satisfaction questions were added, which were set as an external criterion. The answers of the above questionnaires were in the form of 7-point Likert scale. The SERVQUAL questionnaire proved to be very reliable (0.966). All dimensions of the questionnaire evaluating internal quality were rated low by health professionals, with the exception of tangibles. Statistically significant differences were found among some of the dimensions and age of groups, occupation and years of experience. Older and more experienced employees rated internal quality higher. Regarding occupation, differences were found between nursing and administrative staff as well as medical staff. A notable finding is the degree of correlation between all dimensions of internal quality and the job satisfaction questions. As job satisfaction increases, internal quality increases as well and vice versa. Internal quality within an organization and its relationship with job satisfaction, even when it is not the unique requirement for efficient service provision, is a powerful tool for future improvement of health services. Those managers of health care organizations who will be able to identify the factors that determine the welfare and satisfaction of health professionals will derive the best possible performance of hospital staff.