Isoflavones have been suggested to play a role in disease prevention. The accuracy of assessing exposure to isoflavones might be improved by using them as biomarkers. A systematic review of observational studies on the association of isoflavone biomarkers with the risk of chronic disease and mortality was conducted. Meta-analyses of specific biomarker and disease combinations were performed. PubMed and Web of Science databases were searched. Two authors screened the titles and abstracts of candidate publications. The third author was consulted to resolve discrepancies. Forty studies were included and their quality assessed. PRISMA-P guidelines were followed. Eight different isoflavone biomarkers were investigated in association with cancer (26 studies), mortality (2 studies), cardiovascular disease (3 studies), metabolic syndrome risk factors (7 studies), and other outcomes (2 studies). Meta-analyses of studies on individual isoflavonic compounds were conducted for breast and prostate cancer and type 2 diabetes. Higher daidzein and genistein concentrations were associated with lower risk of breast cancer and diabetes. Only daidzein concentrations were inversely associated with risk of prostate cancer. For the remaining endpoints, evidence for associations was inconsistent and scarce, perhaps owing to heterogeneity in study exposures and outcomes. Further research using biomarker information is warranted.