A major challenge for negative-index acoustic metamaterials is increasing their operational frequency to the MHz range in water for applications such as biomedical ultrasound. Herein, a novel technology to realize acoustic metamaterials in water using microstructured silicon chips as unit cells that incorporate silicon nitride membranes and Helmholtz resonators with dimensions below 100 μm fabricated using clean-room microfabrication technology is presented. The silicon chip unit-cells are then assembled to form periodic structures that result in a negative-index metamaterial. Finite-element method (FEM) simulations of the metamaterial show a negative-index branch in the dispersion relation in the 0.25–0.35 MHz range. The metamaterial is characterized experimentally using laser-doppler vibrometry, showing opposite phase and group velocities, a signature of negative-index materials, and is in close agreement with FEM simulations. The experimental measurements also show that the magnitude of phase and group velocities increase as the frequency increases within the negative-index band, confirming the negative-index behavior of the material. Acoustic indices from –1 to –5 are reached with respect to water in the 0.25–0.35 MHz range. The use of silicon technology microfabrication to produce acoustic metamaterials for operation in water opens a new road to reach frequencies relevant for biomedical ultrasound applications.