It is well known that paraoxonase-1 (PON1) activity may decrease during the course of infection and inflammation. The aim of this study was to investigate serum PON1 activity, oxidative status, and thiols levels in patients with acute brucellosis. In addition, we investigated the PON1 phenotype in patients with acute brucellosis. Thirty patients with acute brucellosis and 35 healthy controls were enrolled. Serum paraoxonase and arylesterase activities, thiols levels, lipid hydroperoxide levels, total antioxidant capacity (TAC), total oxidant status (TOS) and oxidative stress index (OSI) were determined. Serum basal and salt-stimulated paraoxonase–arylesterase activities, TAC levels and thiols levels were significantly lower in patients with acute brucellosis than controls (for all, p < 0.05), while LOOH levels, TOS levels, and OSI values were significantly higher (for all, p < 0.05). We concluded that oxidative stress is increased, while serum PON1 activity is decreased in patients with acute brucellosis. These results indicate that lower PON1 activity is associated with oxidant–antioxidant imbalance.