Background: The objective of this study was to investigate how medical and nursing staff of the Nicosia General Hospital is affected by specific motivation factors, and the association between job satisfaction and motivation. Furthermore, to determine the motivational drive of socio-demographic and job related factors in terms of improving work performance. Methods: A previously developed and validated instrument addressing four work-related motivators (job attributes, remuneration, co-workers and achievements) was used. Two categories of health care professionals, medical doctors and dentists (N = 67) and nurses (N = 219) participated and motivation and job satisfaction was compared across socio-demographic and occupational variables. Results: The survey revealed that achievements was ranked first among the four main motivators, followed by remuneration, co-workers and job attributes. The factor remuneration revealed statistically significant differences according to gender, and hospital sector, with female doctors and nurses and accident and emergency (A+E) outpatient doctors reporting greater mean scores (p < 0.005). The medical staff showed statistically significantly lower job satisfaction compared to the nursing staff. Surgical sector nurses and those >55 years of age reported higher job satisfaction when compared to the other groups. Conclusions: The results are in agreement with the literature which focuses attention to management approaches employing both monetary and non-monetary incentives to motivate health care professionals. Health care professionals tend to be motivated more by intrinsic factors, implying that this should be a target for effective employee motivation. Strategies based on the survey's results to enhance employee motivation are suggested.