All healthcare workers (HCWs) encounter stress during in their working lives, and are constantly exposed to adverse conditions. The present study evaluates the relationship between burnout syndrome, anxiety levels and insomnia severity among healthcare workers, who mostly work in shifts. The Maslach Burnout Inventory, the Insomnia Severity Index and the Beck Anxiety Inventory were used to measure burnout, insomnia severity and anxiety status, respectively. This cross sectional study included a total of 1,011 HCWs and 679 (67.2%) of the study respondents were women. The respondents were aged 20-72, with a mean age of 35.67 +/- 8.61 years. Fifty-eight percent (n = 589) of the participants were rotating shift workers. Working on-call led to a significant difference in all burnout parameters (for each, <0.001). Age and on-call duty were seen to lead to a significant difference in the severity of insomnia (p = 0.028, p < 0.001, respectively). The total ISI score was found to be statistically significant positively correlated with the MBI subscales and the total BAI score (for each, <0.001). An increased awareness of the impact of sleep deprivation, burnout and anxiety among HCWs and meaningful interventions promoting change within the healthcare system are needed.