Etiology of acute undifferentiated febrile syndrome (AUFS) is often unknown, leading to inaccurate diagnosis and treatment. Villeta town has been identified as an endemic area for spotted fever group (SFG) rickettsioses but little is known about possible amplifier hosts and other Rickettsia species different from Rickettsia rickettsii. Besides, few studies have approached other AUFS etiologies in the region. We investigated the role of dengue, leptospirosis, rickettsioses, human anaplasmosis, and Q fever as possible causes of AUFS in patients from Villeta. Sera specimens and ticks from animals as well as ticks from vegetation were studied for the presence of different Rickettsia spp. Among 104 sera from patients with AUFS, 16.4%, 24.0%, and 2.9% patients seroconverted to dengue, Leptospira, and SFG Rickettsia, respectively, with a case of probable coinfection or cross-reaction with Anaplasma phagocytophilum. None of the samples were reactive for Coxiella burnetii. Sera samples from 74 horses, 118 dogs, and 62 bovines were collected and showed 33.8%, 14.4%, and 50.0% of seroprevalence for SFG Rickettsia, respectively. A total of 1,287 ixodid ticks were collected from animals/vegetation and processed in pools for polymerase chain reaction. Among them, 1.7% was positive for Rickettsia genes, and Rickettsia amblyommii, R. rickettsii, and Rickettsia spp. were found. These results confirm the circulation of dengue, different SFG Rickettsia species and the relevance of other etiologies like leptospirosis and human anaplasmosis. Further studies must identify different epidemiological variables to establish proper surveillance and control programs.