Due to environmental and ecological changes and suitable habitats, the occurrence of vector-borne diseases is increasing. We investigated the seroprevalence of four major vector-borne pathogens in human patients with febrile illness who were clinically suspected of having Scrub Typhus (ST) caused byOrientia tsutsugamushi. A total of 187 samples (182 patient whole blood and sera samples, including 5 follow-up) were collected. Antibodies toAnaplasma phagocytophilum,Ehrlichia chaffeensis,Borrelia burgdorferi, andBartonella henselaewere tested by using indirect immunofluorescence assays. Molecular diagnoses were performed using real-time PCR. Of the 182 cases, 37 (20.3%) cases were designated as confirmed cases of ST, and the remaining 145 (79.7%) cases as other febrile diseases (OFDs). The seroprevalence ofA.phagocytophilum,E.chaffeensis,B.burgdorferi, andB.henselaewas 51.4% (19/37), 10.8% (4/37), 86.5% (32/37), and 10.8% (4/37) among the ST group, and 42.8% (62/145), 10.4% (19/145), 57.7% (105/145), and 15.9% (29/145) among the OFD group, respectively. There were no significant differences in the seroprevalence between the ST and the OFD groups. Considering the co-occurrence, 89.0% (162/182) had at least one antibody to tick-borne pathogens, 37.0% (60/162) were positive for two pathogens, 17.3% (28/162) for three pathogens, and 6.2% (10/162) for four pathogens. In real-time PCR,O.tsutsugamushiwas positive in 16 cases [15 (40.5%) in ST group and 1 (2.2%) in OFD group], and the four other pathogens were negative in all cases except one confirmed as anaplasmosis. In evaluating the five follow-up samples, the appearance of new antibodies or an increase in the pre-existing antibody titers was detected. Our data highlighted that acute febrile illness and manifestations suggestive of a vector-borne infection must be recognized and further considered for coinfections in clinical practice and the laboratory.