miR-27a-3p targets several proteins on the coagulation cascade. The potential effect of direct oral anticoagulants (DOACs) treatment on miR-27a-3p expression and their broader regulative effect on anticoagulation is unknown. Fifty-nine atrial fibrillation patients treated with rivaroxaban (n = 19), apixaban (n = 27) or dabigatran (n = 13), were included in the study. miR-27a-3p expression was analyzed at baseline and after 7 days of DOAC therapy by using a predesigned TaqMan assay. Relative quantitation of miR-27a-3p expression was calculated and compared in pooled population and in different sample groups. DOAC therapy did not alter miR-27a-3p expression (0.80 fold-change, p = 0.486, pooled population; 0.839 fold-change, p = 0.706, rivaroxaban; 0.921 fold-change, p = 0.800, apixaban; 0.733 fold-change, p = 0.540, dabigatran). miR-27a-3p expression did not differ between controls and bleeding cases (0.833 fold-change, p = 0.588, baseline). Female patients had a trend towards increased baseline expression (1.564 fold-change, p = 0.177) and reduced expression after DOAC treatment (0.683 fold-change, p = 0.243) compared to male patients. Despite the regulatory role of miR-27a-3p on coagulation cascade, treatment with DOACs did not alter its expression. However, additional studies in different ethnic groups are necessary to fully elucidate the effect, if any, of DOACs on miR-27a-3p expression.