To evaluate co-infection of Strongyloides stercoralis and Trypanosoma cruzi and to assess eosinophilia as a screening test for the detection of S. stercoralis infection in patients with Chagas disease (CD). methods A retrospective diagnostic validation study was performed on serum samples from primary care patients diagnosed with CD in the southern Barcelona metropolitan area. All samples with eosinophilia (n = 87) and a random sample of non-eosinophilic sera (n = 180) were selected. Diagnosis of CD was based on positive serology by means of two tests: ORTHO T. cruzi ELISA test, and BIO-FLASH Chagas or Bioelisa CHAGAS. SCIMEDX ELISA STRONGY-96 was used to diagnose strongyloidiasis. results Strongyloides stercoralis serology was positive in 15% of patients of whom 95% showed eosinophilia, vs. 21% of those with negative serology (P < 0.001), with differences in the mean eosinophil count (0.49 vs. 0.27 9 109/l). Only 1.1% of patients with CD but without eosinophilia presented positive serology for S. stercoralis, whereas 44% of patients with CD and eosinophilia did (P < 0.001). Sensitivity and specificity values for eosinophilia were thus 95% and 79%, respectively. PPV was 42.5% and NPV, 98.9%. conclusions The prevalence of co-infection by T. cruzi and S. stercoralis is not negligible and has probably been underestimated for years in many areas, due to frequently subclinical infections. Therefore, serology seems mandatory for these patients and the use of eosinophilia as initial screening could facilitate the task, decreasing the number of analyses to be performed.