Background: At present smoking is considered a great health-related problem. Smoking cigarettes and use of tobacco are on the rise in the Middle East countries; therefore, the number of people exposed to passive cigarette smoke is increasing, too. The aim of the present study was to determine and compare salivary cotinine levels in hookah smokers, individuals exposed to passive cigarette smoke and non-smoker (passive smokers). Methods: In the present cross-sectional study, unstimulated salivary samples were collected from 150 subjects, including 50 hookah smokers, 50 passive smokers, and 50 non-smokers. Bioassay Technology Laboratory cotinine kit was used to determine salivary levels of cotinine using the enzyme-linked immunosorbent assay (ELISA) technique at a sensitivity rate of 0.019 pg/ml. Data were analyzed with SPSS software using t-test and Pearson’s correlation coefficient. Findings: The highest salivary cotinine levels were recorded in hookah users (20.24 ± 5.62 ng/ml), followed by passive smokers (16.09 ± 3.51 ng/ml), in descending order. No detectable cotinine levels were observed in non-smokers. Pearson’s correlation coefficient showed a strong and positive correlation between use of hookah and salivary cotinine levels (r = 0.932, P = 0.001). Conclusion: Based on the results of the present study, salivary cotinine levels were higher in hookah smokers compared with passive smokers and non-smokers, in descending order.