This paper addresses some of the existing research gaps in the practical use of acoustic waves for navigation of autonomous air and surface vehicles. After providing a characterisation of ultrasonic transducers, a multistatic sensor arrangement is discussed, with multiple transmitters broadcasting their respective signals in a round-robin fashion, following a time division multiple access (TDMA) scheme. In particular, an optimisation methodology for the placement of transmitters in a given test volume is presented with the objective of minimizing the position dilution of precision (PDOP) and maximizing the sensor availability. Additionally, the contribution of platform dynamics to positioning error is also analysed in order to support future ground and flight vehicle test activities. Results are presented of both theoretical and experimental data analysis performed to determine the positioning accuracy attainable from the proposed multistatic acoustic navigation sensor. In particular, the ranging errors due to signal delays and attenuation of sound waves in air are analytically derived, and static indoor positioning tests are performed to determine the positioning accuracy attainable with different transmitter–receiver-relative geometries. Additionally, it is shown that the proposed transmitter placement optimisation methodology leads to increased accuracy and better coverage in an indoor environment, where the required position, velocity, and time (PVT) data cannot be delivered by satellite-based navigation systems.