Background: Job satisfaction is an important condition for staff retention in most healthcare Organizations. As a concept, job satisfaction is linked to motivation theory. Herzberg's two factor theory of motivation is used in this study to explore what motivational elements are associated with job satisfaction among medical laboratory professionals (MLPs) in Oman. Methods: A mixed-method approach was adopted, and focus group discussions (FGDs) were used for data collection. The FGDs were conducted in the main hospitals in Oman. Data were analyzed by directed content analysis, and frequencies of statements related to factors were calculated for a comparison with the Herzberg theory. Results: The following job dissatisfaction factors (hygiene) were identified: health and safety, heavy workload, salary, promotion, recognition and organizational policies. The satisfaction (motivators) were: relationships with co-workers, relationship with leaders, and professional development. Conclusions: The job dissatisfaction reported was resulted from the absence of hygiene factors and some of the motivators in accordance with Hertzberg's theory. Hospital managers need to address these factors, defined by Hertzberg, in order to improve motivation and job satisfaction.