Prevention of transfusion-related Chagas disease in Mexico City depends on targeted questionnaire-based screening of donors by nurses at blood banks. To assess potential problems with this strategy, surveys were distributed to the nurses who screen donors in a random sampling of nine blood banks in Mexico City, to measure appropriate knowledge about Chagas disease. We found that 80% (95% CI 68-92%) of nurses answered at least one of the three donor risk factor questions incorrectly, which may fail to trigger confirmatory laboratory testing of potentially infected units. If this knowledge deficit is widespread, up to 680,000 units (95% CI 578,000-782,000 units) of donated blood could be potentially contaminated with Chagas disease in Mexico. In place of targeted screening, routine laboratory testing of all donated blood would be a cost-effective method to safeguard blood recipients from iatrogenic Chagas disease.