Brucellosis is a zoonosis of great and worldwide public health concern that can cause a severe febrile illness in humans. In Pakistan, brucellosis is a critical problem in both animals and humans. This study aimed to gain insight into its prevalence and to analyze the potential risk factors of patients with acute febrile illness (AFI) of an unknown cause, at the hospitals of Rawalpindi and Islamabad in Pakistan. In total, 446 blood samples were collected from patients and screened for brucellosis using the Rose Bengal Plat Test (RBPT). All the serum samples were investigated for Brucella DNA using specific real-time PCR. Age, sex, occupation, urbanicity, socioeconomic status and history of animal contact were recorded and assessed as potential risk factors. The proportion of acute febrile illness patients for whom brucellosis could be suspected was 10.1% by the RBPT. Brucella DNA was detected in 26 (5.8%) cases and identified as B. abortus. Contact with infected animals, consumption of raw milk and socioeconomic status showed a highly significant (p < 0.05) correlation with seropositivity. Elderly patients (19.7% RBPT and 12.1% PCR) and females (13% RBPT and 9.3% PCR) were of high risk of brucellosis. Patients suffering from brucellosis-related manifestations should be screened for brucellosis, especially those in contact with animals or those consuming their unprocessed products, given the increased risk. The results of this study, which highlight that Brucella abortus as an important cause of acute febrile illnesses in humans, aid the development of effective control strategies for human brucellosis in Pakistan.