Due to the limited output capability of piezoelectric diaphragm pumps, the driving voltage is frequently increased to obtain the desired output. However, the excessive voltage application may lead to a large deformation in the piezoelectric ceramics, which could cause it to breakdown or become damaged. Therefore, increasing the number of chambers to obtain the desired output is proposed. Using a check-valve quintuple-chamber pump with quintuple piezoelectric actuators, the characteristics of the pump under different driving modes are investigated through experiments. By changing the number and connection mode of working actuators, pump performances in terms of flow rate and backpressure are tested at a voltage of 150 V with a frequency range of 60 Hz -400 Hz. Experiment results indicate that the properties of the multiple-chamber pump change significantly with distinct working chambers even though the number of pumping chambers is the same. Pump performance declines as the distance between the working actuators increases. Moreover, pump performance declines dramatically when the working piezoelectric actuator closest to the outlet is involved. The maximum backpressures of the pump with triple, quadruple, and quintuple actuators are increased by 39%, 83%, and 128%, respectively, compared with the pump with double working actuators; the corresponding maximum flow rates of the pumps are simply increased by 25.9%, 49.2%, and 67.8%, respectively. The proposed research offers practical guidance for the effective utilization of the multiple-chamber pumps under different driving modes.