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The purpose of this study was to assess the effect of chemotherapy on brain functional resting-state signal variability and cognitive function in older long-term survivors of breast cancer. This prospective longitudinal study enrolled women age ≥ 65 years of age who were breast cancer survivors after exposure to chemotherapy (CH), age-matched survivors not exposed to chemotherapy, and healthy controls. Participants completed resting-state functional brain MRI and neurocognitive testing upon enrollment (timepoint 1, TP1) and again two years later (timepoint 2, TP2). There were 20 participants in each of the three groups at TP1. The CH group showed a significant decrease in SD_BOLD (blood-oxygen-level-dependent signal variability in standard deviation) in the right middle occipital gyrus (ΔSD_BOLD = −0.0018, <i>p</i> = 0.0085, q (p_FDR) = 0.043 at MNI (42, −76, 17)) and right middle temporal gyrus (ΔSD_BOLD = −0.0021, <i>p</i> = 0.0006, q (p_FDR) = 0.001 at MNI (63, −39, −12)). There were negative correlations between the crystallized composite scores and SD_BOLD values at the right inferior occipital gyrus (correlation coefficient r = −0.84, <i>p</i> = 0.001, q (p_FDR) = 0.016) and right middle temporal gyrus (r = −0.88, <i>p</i> = 0.000, q (p_FDR) = 0.017) for the CH group at TP1. SD_BOLD could be a potentially useful neuroimaging marker for older long-term survivors of breast cancer with exposure to chemotherapy.