The management of pelvic floor dysfunctions might need to be based on a comprehensive neuro-musculoskeletal therapy such as The Rolf Method of Structural Integration (SI). The aim of the study was to evaluate the pelvic floor muscle (PFM) after the tenth session of SI by using surface electromyography (sEMG). This was a randomized, interventional study. Thirty-three healthy women were randomly assigned to the experimental (SI) or control group. The outcome measures included PFM bioelectrical activity, assessed using sEMG and endovaginal probes. An intervention in the SI group included 60 min of SI once a week, and teaching on how to contract and relax PFMs; in the control group, only the teaching was carried out. In the SI group, a significant difference was found between the PFM sEMG activity during “pre-baseline rest” (p < 0.014) and that during “rest after tonic contraction” (p = 0.021) in the supine position, as were significant increases in “phasic contraction” in the standing position (p = 0.014). In the intergroup comparison, higher PFM sEMG activity after the intervention “phasic contraction” (p = 0.037) and “pre-baseline rest” (p = 0.028) was observed in the SI group. The SI intervention significantly changes some functional bioelectrical activity of PFMs, providing a basis for further research on a new approach to PFM facilitation, particularly in clinical populations.