Purpose Studies examining biomarkers associated with fatigue in breast cancer survivors treated with radiation are limited. Therefore, we examined the longitudinal association between serum biomarkers and post-breast cancer fatigue in survivors treated with radiation: [oxidative stress] 8-hydroxyguanosine, myeloperoxidase; [inflammation] interleukin-6 (IL-6), c-reactive protein, growth differentiation factor-15 (GDF-15), placental growth factor, transforming growth factor-beta, [cardiac damage] cystatin-C, troponin-I. Methods In a secondary analysis, we included participants from the Women’s Health Initiative if they had: a previous breast cancer diagnosis (stages I-III), no prior cardiovascular diseases, pre-and post-breast cancer serum samples drawn approximately 3 years apart, and fatigue measured using the Short-Form 36 vitality subscale at both serum collections. Biomarkers were measured using ELISA or RT-qPCR and modeled as the log₂ post-to pre-breast cancer ratio. Results Overall, 180 women with a mean (SD) age of 67.0 (5.5) years were included. The mean (SD) vitality scores were 66.2 (17.2) and 59.7 (19.7) pre- and post-breast cancer, respectively. Using multivariable weighted linear regression, higher biomarker ratios of cystatin-C, IL-6, and GDF-15 were associated with a lower vitality score (i.e., higher fatigue). For example, for each 2-fold difference in cystatin-C biomarker ratio, the vitality score was lower by 7.31 points (95% CI: −14.2, −0.45). Conclusion Inflammatory and cardiac damage biomarkers are associated with fatigue in breast cancer survivors treated with radiation; however, these findings should be replicated in a larger sample. Biomarkers could be measured in clinical practice or assessed in risk prediction models to help identify patients at high risk for fatigue.