Background Acute febrile illnesses (AFIs), including dengue, scrub typhus and leptospirosis, cause significant morbidity and mortality in Southeast Asia. Serological surveillance can be used to investigate the force and distribution of infections. Dried blood spot (DBS) samples are an attractive alternative to serum because they are easier to collect and transport and require less cold storage. We conducted a pilot study to determine the feasibility of integrating serological surveillance for dengue, scrub typhus and leptospirosis into a population-representative lymphatic filariasis seroprevalence survey in Timor-Leste using DBSs. Methods A total of 272 DBSs were collected from healthy community participants. DBSs were analysed at the National Health Laboratory using commercially available enzyme-linked immunosorbent assays. To validate assays for DBSs, 20 anonymised serum samples of unknown serostatus were used to create dried serum spots (DSSs). These were analysed with optical densities compared with those of serum. Where low variance was observed (dengue assay) the published kit cut-offs for serum were applied to the analysis of DBSs. For the other assays (scrub typhus and leptospirosis), index values (IVs) were calculated and cut-offs were determined to be at 2 standard deviations (SDs) above the mean. Results Of the 272 samples analysed, 19 (7.0% [95% confidence interval {CI} 4.3 to 10.7]) were positive for dengue immunoglobulin G (IgG), 11 (4.0% [95% CI 2.1 to 7.1]) were positive for scrub typhus IgG and 16 (5.9% [95% CI 3.4 to 9.4%]) were positive for leptospira IgG. Conclusions While dengue seroprevalence was lower than in nearby countries, results represent the first evidence of scrub typhus and leptospirosis transmission in Timor-Leste. Integrated programmes of serological surveillance could greatly improve our understanding of infectious disease epidemiology in remote areas and would incur minimal additional fieldwork costs. However, when planning such studies, the choice of assays, their validation for DBSs and the laboratory infrastructure and technical expertise at the proposed location of analysis must be considered.