We describe the design, fabrication and testing of a biologically-driven actuator which serves as a proof-or-concept “artificial heartbeat” for future use within bio-robotic art and design. The actuator employs live biological material, both as a source of power and means of actuation. Pneumatic pressure generated by the action of the yeast Saccharomyces cerevisiae causes a diaphragm to distend. Movement of the diaphragm is regulated by a purpose-built control valve. When the diaphragm is fully distended, the valve opens to release pressure, returning the actuator to its state of rest in readiness for the next actuation cycle.The control valve employs a temperature-responsive NiTi “artificial muscle” which is activated when heated electrically using power generated by microbial fuel cells. In an alternative embodiment, the NiTi valve is powered by solar energy via photovoltaic panels. Results are presented showing the performance of devices powered by both energy sources. The structure of the bio-actuator is fabricated by 3D printing and rapid tooling techniques.Bio-actuation may be employed for such functions as shapechange, pumping and propulsion. Possible applications for the physical principles described in this paper range from energy autonomous robotics and artificial life to artworks which creatively exploit robotic and bio-technology.