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In South Africa, the prevalence of cattle handler exposure to <i>Brucella</i> on cattle farms is unknown and risk factors and cattle symptoms associated with infected cattle herds are unavailable. To address this gap, a case-control study of cattle herds was conducted in Gauteng province and farm workers and veterinary officials were tested for exposure to <i>Brucella</i>. Seroprevalence amongst farm workers exposed to case herds ranged from 4.0% (BrucellaCapt^®) to 16.7% (IgG ELISA^®), compared to those exposed to control herds, where seroprevalence ranged from 1.9% (BrucellaCapt^®) to 5.7% (IgG ELISA^®). Seroprevalence amongst veterinary officials was significantly greater compared to farm workers exposed to case herds for the outcome RBT+ IgM- IgG+ (OR = 11.1, 95% CI: 2.5–49.9, <i>p</i> = 0.002) and RBT- IgM- IgG+ (OR = 6.3, 95% CI: 2.3–17.3, <i>p</i> < 0.001). Risk factors associated with being an infected herd were: being a government-sponsored farm vs. private farm (OR 4.0; 95% CI: 1.4–11.3; <i>p</i> = 0.009), beef vs. dairy herd (OR 7.9; 95% CI: 1.4–44.9; <i>p</i> = 0.020), open vs. closed herd (OR 3.3; 95% CI: 1.1–10.4; <i>p</i> = 0.038) and the presence of antelope on the farm (OR 29.4; 95% CI: 4.0–218.2; <i>p</i> = 0.001). Abortions (OR = 5.1; 95% CI: 2.0–13.3; <i>p</i> < 0.001), weak calves in the herd (OR = 8.0; 95% CI: 2.6–24.4; <i>p</i> < 0.001), reduction in number of calves born (OR = 9.0; 95% CI: 2.1–43.6; <i>p</i> < 0.001), reduction in conception rate (OR = 3.9; 95% CI: 0.8–18.3; <i>p</i> = 0.046), hygromas in cattle (<i>p</i> = 0.011) and farmers reporting brucellosis-like symptoms in their farm workers or in him/herself (OR = 3.4; 95% CI: 1.3–8.7; <i>p</i> = 0.006) were more likely to be associated with <i>Brucella</i> infected herds than control herds. This evidence can be used in strategic planning to protect both human and herd health.