The diagnostic performance of serum carcinoembryonic antigen (CEA) and carbohydrate antigen (CA) 19-9 levels for multiple-organ cancer screening has not been fully elucidated. However, they are widely used for real-world opportunistic screening of multiple-organ cancers. This study aimed to examine the diagnostic performance of these serum markers in multiple-organ cancer screening. Data from asymptomatic individuals subjected to opportunistic cancer screening were analyzed. The diagnostic performance of CEA and CA 19-9 was assessed for (A) upper/lower gastrointestinal cancers and (B) whole-body cancers (including both gastrointestinal and other organ cancers) using the results of upper/lower gastrointestinal endoscopy and whole-body imaging as reference. Data from 12,349 and 7616 screened individuals were used to assess the diagnostic performance of CEA and CA 19-9 for (A) and (B), respectively. For (A), the sensitivity and positive predictive value (PPV) of CEA (cut-off: 5 ng/mL) were 7.8% and 3.7%, respectively; those of CA19-9 (cut-off: 37 U/mL) were 7.4% and 2.7%, respectively. For (B), the sensitivity and PPV of CEA were 6.6% and 4.1%, respectively, and those of CA19-9 were 10.8% and 5.8%, respectively. Considering even multiple cancers, the sensitivity and PPV of CEA and CA 19-9 were low, thus confirming their limited usefulness in multiple-organ cancer screening.