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Background and Aim: Brucellosis is a highly contagious, neglected zoonotic disease of major importance worldwide. The disease is endemic in many countries, burdening healthcare systems and the livestock industry and representing a persistent public health concern in these countries. Brucellosis is considered an important occupational hazard for livestock workers. Limited studies have investigated human brucellosis in Saudi Arabia. Therefore, this study aimed to estimate the prevalence of brucellosis among employees of high-risk brucellosis professions, including veterinarians, animal herders, and abattoir workers in Madinah, Saudi Arabia, and to determine the associated risk factors. Materials and Methods: A cross-sectional study was conducted in Madinah, Saudi Arabia, during the period of January-March 2023. Ninety blood samples were collected from individuals occupationally at risk of exposure to Brucella infections. Serum samples were examined for immunoglobulins (Ig)M and IgG antibodies against Brucella using an indirect enzyme-linked immunosorbent assay. Before sample collection, a predesigned online questionnaire was used to collect the participants' sociodemographic characteristics and the probable risk factors for human brucellosis. A Chi-square test was used to compare the differences among groups; p < 0.05 were considered statistically significant. Results: Among the 90 participants among the high-risk individuals, Brucella IgM and IgG seropositivity were found in 8 (8.8%) and 11 (12.12%) cases, respectively. IgM mono antibody positivity was observed in 4 (4.44%) and 7 (7.77%) of the study population who tested positive for IgG only. Dual positivity for IgM and IgG antibodies was observed in 4 (4.44%) participants. No significant association was determined between seropositivity and age, urbanicity, education, occupation, and duration of exposure (p > 0.05). Conclusion: Brucellosis is a high-risk occupational disease among workers with close contact with livestock. This study demonstrates that the seroprevalence of brucellosis among occupationally high-risk individuals in Madinah, Saudi Arabia, is relatively low compared to other countries in the region. Nevertheless, educational programs should be implemented to improve knowledge regarding brucellosis, particularly among high-risk individuals. [ABSTRACT FROM AUTHOR]