Background Women with symptoms and signs of ischemia, preserved left ventricular ejection fraction (LVEF), and no obstructive coronary artery disease (CAD), often have coronary microvascular dysfunction (CMD), and are at risk of future heart failure with preserved ejection fraction (HFpEF). N-terminal pro-B-type natriuretic peptide (NT-proBNP) is used to evaluate HF and myocardial ischemia. Relationships between NT-proBNP and CMD are not well defined in this population. Methods We evaluated resting NT-proBNP levels in 208 women with symptoms and signs of ischemic heart disease, preserved LVEF and no obstructive CAD undergoing clinically indicated invasive coronary flow reserve (CFR) as a measure of CMD-related ischemia and resting left ventricular end-diastolic pressure (LVEDP). Chi-square testing was used for categorical variables and ANOVA or Kruskal-Wallis tests were used for continuous variables. Results Overall, 79% had an elevated resting LVEDP, and mean NT-proBNP was 115 ± 158 pg/mL. NT-proBNP levels correlated directly with age (r = 0.28, p = <0.0001), and indirectly with body mass index (r = -0.21, p = 0.0006), but did not independently associate with CFR. When stratified by NT-proBNP thresholds, higher NT-proBNP was initially associated with lower CFR, which did not persist with adjustment for multiple testing (p = 0.01 and 0.36, respectively). Conclusion Among women with symptoms and signs of ischemia, preserved LVEF, no obstructive CAD, and undergoing clinically indicated functional coronary angiography (FCA) for suspected CMD, while a majority had elevated resting LVEDP, we failed to find an independent association between CFR and NT-proBNP, although stratified clinical thresholds may relate to lower CFR. Further work is needed to investigate if these findings support the hypothesis that CMD-related ischemia may be a precursor to HFpEF.