Background: Hepatitis B is one of the major causes of mortality among viral diseases. To reduce morbidity rate and increase knowledge of people about potential risk factors, the aim of this study was to determine the prevalence of hepatitis B among the general population and the risk factors associated with hepatitis B virus (HBV) infection in Isfahan, Iran. Materials and Methods: In a case–control study, 314 HBV-infected patients and 557 healthy participants were recruited. Data on demographics, immunization history, medical history, family medical history, life history, therapeutic factors, and behavioral risk factors were collected through a standard checklist. Chi-square and logistic regression were used for univariate and multivariable analyses. Results: Our results showed that among sociodemographic variables, higher age, being male, lower economic status, and lower educational attainments increased the risk of affecting by HBV (odds ratio [OR] >1, P < 0.001); furthermore, Iranian and no immigrant people showed higher significant risk of being affected by HBV. Multivariable logistic regression showed among medical, blood, and behavioral risk factors, family history of hepatitis (OR: 10.56; 95% confidence interval [CI]: 4.56–24.86), dental treatment history (OR: 4.30; 95% CI: 1.41–13.10), and hospitalization (OR: 2.94; 95% CI: 1.72–5.00). Conclusion: Our results demonstrated that there are still several risk factors for hepatitis B surface antigen infection among the Iranian adult population. Immunization programs should continue and focus on high-risk adults, and interventions should be directed toward to reduce risk factors associated with hepatitis B.