An experimental study was conducted to investigate the effects of acoustic waves on the growth of three-dimensional perturbations in a plane mixing layer using a novel experimental technique in a towing tunnel. The mixing layer in the transitional regime at Rem ≈ 100 based on the momentum thickness was investigated by means of flow visualization/flying hot-wire technique. We found that vortex pairing in the mixing layer excited with a subharmonic mode prevents (or delays) the rapid growth of three-dimensionality. In contrast, delayed pairing with a fundamental mode can result in significant growth of three-dimensionality prior to the pairing, where the rollers are bent in the spanwise direction due to the secondary instability of the elliptic vortex. These results are in good agreement with the observations of simulated mixing layers at Rem=112. The mechanisms responsible for the growth of three-dimensionality during pairing are described.