Polymersomes, vesicles composed of block copolymers, are promising candidates as membrane alternatives and functional containers, e.g., as potential carriers for functional molecules because of their stability and tunable membrane properties. In the scope of possible use for membrane protein delivery to cells by electrofusion, we investigated the cytotoxicity of such polymersomes as well as the effects of nanosecond electric pulses with variable repetition rate on the shape and permeability of polymersomes in buffers with different conductivities. The polymersomes did not show cytotoxic effects to CHO and B16-F1 cells in vitro in concentrations up to 250 µg/mL (for 48 h) or 1.35 mg/mL (for 60 min), which renders them suitable for interacting with living cells. We observed a significant effect of the pulse repetition rate on electrodeformation of the polymersomes. The electrodeformation was most pronounced in low conductivity buffer, which is favorable for performing electrofusion with cells. However, despite more pronounced deformation at higher pulse repetition rate, the electroporation performance of polymersomes was unaffected and remained in similar ranges both at 10 Hz and 10 kHz. This phenomenon is possibly due to the higher stability and rigidity of polymer vesicles, compared to liposomes, and can serve as an advantage (or disadvantage) depending on the aim in employing polymersomes such as stable membrane alternative architectures or drug vehicles.