Acute undifferentiated febrile illness (AUFI) is the main cause of medical attention in the tropics worldwide. Malaria, arboviral diseases, and leptospirosis are the most important etiologies. These are highly endemic in the Urabá antioqueño, Colombia, being the main causes of fever in several municipalities in this region. However, up-to-date data regarding the infecting species and serotypes are lacking. Thus, we characterized the etiology of AUFI, focusing on malaria, arboviruses, and leptospirosis in this region and the circulating infecting species. An active surveillance was conducted between January and April 2022, and July and October 2023 in two local hospitals in the Urabá antioqueño. Febrile patients were enrolled voluntarily. Malaria, arboviral diseases, and leptospirosis were screened through direct, serological, molecular, and rapid diagnostic methods. Amplicons obtained for dengue virus (DENV) and Leptospira spp. were analyzed through phylogenetic analysis. A total of 184 febrile patients were enrolled. A confirmed etiology was detected in 43.4% of patients from Apartadó and 61.2% from Turbo. Malaria was the most frequent cause in both municipalities, which was caused mainly by Plasmodium falciparum in Apartadó and Plasmodium vivax in Turbo. Dengue virus serotype 1 genotype V, DENV genotype Asian-American, and DENV genotype Cosmopolitan were identified, as well as pathogenic Leptospira species closely related to Leptospira santarosai and Leptospira noguchii. The present study confirms the importance of malaria, dengue fever, and leptospirosis in the Urabá antioqueño. Plasmodium falciparum and P. vivax were identified, as well as two DENV serotypes and three DENV genotypes and two different Leptospira species.