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Background The etiology and optimal clinical management of acute febrile illness (AFI) is poorly understood. Methods Blood samples taken from study participants with acute fever (≥37.5°C) or a history of fever and recruited into the previous Typhoid Fever Surveillance in Africa (TSAP) study were evaluated using a polymerase chain reaction (PCR)-based TaqMan-Array Card designed to detect a panel of bacterial, viral, and parasitic pathogens. Clinical metadata were also assessed. Results A total of 615 blood samples available for analysis originated from Burkina Faso (n = 53), Madagascar (n = 364), and Sudan (n = 198) and were taken from participants ranging in age from 0–19 years. Through the TaqMan-Array Card, at least 1 pathogen was detected in 62% (33 of 53), 24% (86 of 364), and 60% (118 of 198) of specimens from Burkina Faso, Madagascar, and Sudan, respectively. The leading identified pathogen overall was Plasmodium spp. accounting for 47% (25 of 53), 2.2% (8 of 364), and 45% (90 of 198) of AFI at the respective sites. In Madagascar, dengue virus was the most prevalent pathogen (10.2%). Overall, 69% (357 of 516) of patients with clinical diagnoses of malaria, respiratory infection, or gastrointestinal infection were prescribed a World Health Organization guideline-recommended empiric antibiotic, whereas only 45% (106 of 237) of patients with pathogens detected were treated with an antibiotic exerting likely activity. Conclusions A PCR approach for identifying multiple bacterial, viral, and parasitic pathogens in whole blood unveiled a diversity of previously undetected pathogens in AFI cases and carries implications for the appropriate management of this common syndrome. [ABSTRACT FROM AUTHOR]