Introduction: Vaginal dysbiosis affects pregnancy outcomes, however, the relevance of abnormal findings on pre/post-surgical vaginal culture in women undergoing fetal spina bifida (fSB) repair is unknown. Objectives: To describe the incidence of normal and abnormal pre- and post-surgical vaginal microorganisms in fSB patients and to investigate potential associations between the type of vaginal flora and the occurrence of preterm prelabour rupture of membranes (PPROM) and preterm birth (PTB). Methods: 99 women undergoing fSB repair were eligible (2010–2019). Pre-surgical vaginal culture was routinely taken before surgery. Post-surgical cultures were taken on indication. Vaginal flora was categorized into four categories: healthy vaginal flora (HVF), bacterial vaginosis (BV), desquamative inflammatory vaginitis (DIV), and yeast infection. Results: The incidence of HVF, BV, DIV, or yeast infections was not statistically different between the pre- and postoperative patients. Furthermore, an abnormal pre/post-surgical vaginal flora was not associated with PPROM (OR 1.57 (0.74–3.32), p = 0.213)/OR 1.26 (0.62–2.55), p = 0.515), or with PTB (OR 1.19 (0.82–1.73), p = 0.315)/(OR 0.86 (0.60–1.24), p = 0.425). Conclusions: Abnormal vaginal microbiome was not associated with PPROM and PTB when appropriate treatment was performed.