We conducted a longitudinal observational study of 533 patients presenting to two hospitals in the Ecuadorean Amazon basin with acute undifferentiated febrile illness (AUFI) from 2001 through 2004. Viral isolation, reverse transcription-polymerase chain reaction (RT-PCR), IgM seroconversion, and malaria smears identified pathogens responsible for fever in 122 (40.1%) of 304 patients who provided both acute and convalescent blood samples. Leptospirosis was found in 40 (13.2%), malaria in 38 (12.5%), rickettsioses in 18 (5.9%), dengue fever in 16 (5.3%), Q fever in 15 (4.9%), brucellosis in 4 (1.3%), Ilhéus infection in 3 (1.0%), and Venezuelan equine encephalitis (VEE), Oropouche, and St. Louis encephalitis virus infections in less than 1% of these patients. Viral isolation and RT-PCR on another 229 participants who provided only acute samples identified 3 cases of dengue fever, 2 of VEE, and 1 of Ilhéus. None of these pathogens, except for malaria, had previously been detected in the study area.