Passive air-breathing microbial fuel cells (MFCs) are a promising technology for energy recovery from wastewater and their performance is highly dependent on characteristics of the separator that isolates the anaerobic anode from the air-breathing cathode. The goal of the present work is to systematically study the separator characteristics and its effect on the performance of passive air-breathing flat-plate MFCs (FPMFCs). This was performed through characterization of structure, properties, and performance correlations of eight separators in Part 1 of this work. Eight commercial separators were characterized, in non-inoculated and inoculated setups, and were examined in passive air-breathing FPMFCs with different electrode spacing. The results showed a decrease in the peak power density as the oxygen and ethanol mass transfer coefficients in the separators increased, due to the increase of mixed potentials especially at smaller electrode spacing. Increasing the electrode spacing was therefore desirable for the application of diaphragms. The highest peak power density was measured using Nafion®117 with minimal electrode spacing, whereas using Nafion®117 or Celgard® with larger electrode spacing resulted in similar peak powers. Part 2 of this work focuses on numerical modelling of the FPMFCs based on mixed potential theory, implementing the experimental data from Part 1.