The effect of modifying the geometrical configuration of a co-linear chamber to improve its performance and to increase the microbial inactivation was investigated numerically and validated experimentally. In non-thermal radio frequency electric fields (RFEF) processing, co-linear chambers exhibit low tendency to dielectric breakdown and arcing, especially in liquid food products with high electrical conductivities, but they show low energy efficiency and poor uniformity of processing conditions. Accordingly, a standard co-linear chamber (C1) with 1 mm of length and 3 mm of diameter was selected. The chamber included a gap between the end of electrodes and the treatment zone, as well as a diameter contraction at that zone. The geometry of the standard co-linear chamber was modified by removing the recirculation/stagnation zones adding stainless steel tubes (C2), as well as through the additional insertion of stainless steel mesh to contain the electric field within the treatment zone (C3). These configurations were evaluated numerically using COMSOL Multiphysics modelling. The model was validated by comparing experimental measurements of outlet temperature and power consumption with the model predictions. The numerical study showed that C3 exhibited a more uniformly distributed electric field and temperature profiles as well as higher velocities and turbulent kinetic energy that were also more evenly distributed within the treatment zone, compared to other configurations. In experimental studies, C3 achieved the highest microbial inactivation at constant values of peak voltage, electric field strength, and energy levels. Furthermore, C3 showed the most energy efficiency among the three geometrical configurations. Industrial relevance As an alternative to thermal processing, radio frequency electric fields (RFEF) processing needs to ensure the microbial safety of the food products in an energy-efficient manner due to the challenges on controlling Joule heating, which can lead to arcing and the dielectric breakdown of the treatment chamber. This can be achieved by designing treatment chambers that can enclose the electric field within the treatment zone while providing a more evenly distributed electric field, hydrodynamic and temperature profiles within that zone. This study proves that the geometrical configuration of co-linear chambers can be altered to minimize the energy consumption while maximizing the microbial inactivation. The findings can be used for scaling up and advancing the industrial application of RFEF technology for food processing.