To investigate the prevalence and risk factors of brucellosis in human and animal's communities in southern Saudi Arabia. Methods: A cross-sectional sero-epidemiological study was conducted in Aseer and Jazan, Saudi Arabia (October 2017-October 2018). Human serum samples (n=339) were initially screened for Brucella antibodies and positive samples were further titrated for Brucella antibodies by immunocapture assay (titer of 1:320 as positive). Animal samples (n=828) were screened using the Rose Bengal test. Relationship status was dichotomized to measure and predict independent contributions to variations in human using univariate and multivariate stepwise binary logistic regression model. Results: The rate of brucellosis among the 339 human samples in the two regions was 33.9%, and the rate of acute brucellosis was 12.4%. The rate of brucellosis in animals was 4.7%. Human brucellosis among the target groups was higher in northwestern Aseer (53.3%) compared to Southeastern Aseer (25.9%) and Jazan region (20.6%). The disease was more prevalent among non-Saudi nationals (35.2%) compared to Saudis (30.5%). The rate of brucellosis among butchers and shepherds was 37.5% and 37.2%, respectively. The rate of brucellosis was 37.8% in people over 30 years of age. Our univariate analysis showed that residing in Aseer region (OR: 2.60, 95% CI: 1.50-4.40), especially residing in northwestern Aseer region (OR: 4.40, 95% CI: 2.40-7.90), frequent consumption of raw meat (OR: 2.90, 95% CI: 1.50-5.50), shepherds (OR: 2.10, 95% CI: 0.80-5.30), owning sheep (OR: 2.20, 95% CI: 1.10-4.40), daily contact with animals (OR: 2.10, 95% CI: 0.75-5.80), and those > 30-year-old (OR: 1.50, 95% CI: 1.00-2.40) were significantly associated with increased risks of brucellosis. Our multivariate analysis further showed that residing in northwestern Aseer (OR: 9.16, 95% CI: 3.39-24.76) and having sheep (OR: 1.16, 95% CI: 1.00-1.35) were significant and independent risks of brucellosis while residing in agricultural region (OR: 0.28, 95% CI: 0.10-0.78) was a significant and independent protector against brucellosis. Conclusions: The study concluded that residing in northwestern Aseer area and having animals (sheep) are associated with significantly increased risks of brucellosis.