Background: Certain occupational groups, particularly barbers and butchers, face an increased risk of hepatitis B virus (HBV) infection. HBV is a partially double-stranded DNA virus from Hepadnaviridae family and a major global health concern for occupational health professionals due to their frequent contact with sharp instruments and the potential for blood borne pathogen transmission. Also The virus can remain infectious on environmental surfaces for seven days or more and has a very low minimum infectious dose, making contaminated razors and knives effective vectors of infection. This study aimed to determine the prevalence of hepatitis B surface antigen (HBsAg) positivity among barbers and butchers in Salah al-Din Governorate, Iraq, and compare it to a control group that had not been exposed to the virus. It also aimed to describe some demographic and occupational characteristics of participants infected with HBsAg using a survey. Method: A cross-sectional study conducted in Salah Al-Din Governorate, Iraq, specimen collected from January to June 2025. The study included 900 specimen with 20–70 years in age, encompassed 300 Barbers, 300 Butchers, and 300 healthy individuals as a control group. Demographic and occupational data were collected through survey, while Serum samples were tested for hepatitis B surface antigen (HBsAg) using ELISA, with statistical significance considered at P < 0.05. Results: This study included 900 samples, equally divided into 300 samples each for barbers, butchers, and a control group. A positive result of (HBsAg) appearance in 34 (11.3%) barbers and 28 (9.3%) butchers, while no positive cases in the control group. The prevalence of hepatitis B infection differed significantly among the three study groups (χ² = 34.229, df = 2, p < 0.001). Among barbers, infection was significantly associated with age, with the highest prevalence observed among participants aged 31–40 years (20.0%). In contrast, no significant association between age and hepatitis B infection was found among butchers. Notably, most HBsAg-positive participants did not report any symptoms, indicating that infection was frequently asymptomatic. Occupational factors also showed a considerable risk of exposure, with accidental cuts reported more frequently among butchers (71.4%). In addition, the methods used to decontaminate and sterilize working tools varied between barbers and butchers, reflecting differences in occupational hygiene practices Conclusion: The results showed that the prevalence of hepatitis B surface antigen (HBsAg) was higher among barbers compared to butchers. In addition, most HBsAg infections were asymptomatic. These results show the need to target occupational health professionals more through hepatitis B vaccination and continuous periodic screening, along with taking infection control measures in these professions specifically, given their importance in increasing the spread of the disease among other groups. Further studies should be conducted by taking samples from different locations and performing confirmatory molecular tests.