Cultivation-based and DNA-based methods for determining the bacterial load and the composition of the bacterial spectrum have been successfully established for media in electrodip painting, and used for the detailed analysis of the contamination situation in an E-coating system of an automobile plant in Germany. Dominating representatives of the genus Microbacterium spp., the orders Burkholderiales and Pseudomonadales, the family Cytophagaceae and the genera Corynebacterium spp., Sphingomonas spp., and Stenotrophomonas spp. were used for inactivation experiments. Different pulsed electric field (PEF) parameters were studied for an effective and target-directed inactivation of defined bacterial suspensions containing mixtures of Gram-positive as well as Gram-negative bacteria, but also single species suspensions in adequate liquids. PEF treatment with pulse durations longer than 1.0 µs effectively killed bacteria even in low conductivity media, regardless of whether the pulses were unipolar or bipolar, indicating that the choice of pulse shape does not limit the design of the PEF system. Model calculations showed that for efficient treatment in bypass mode, a high treatment flow rate is required rather than a high inactivation efficiency of the PEF treatment. By using specific treatment parameters, such as bipolar pulses of 50 k Vcm−1 and a treatment energy of 40 J mL−1, a significant reduction in both Gram-negative and Gram-positive bacteria (> 2 log10 reduction) can be achieved while minimizing electrode corrosion and coating degradation. PEF treatment proves to be an effective alternative to the use of biocides in an E-coating system and can help maintain a bacteriostatic environment in the system by operating at different points, in transfer flow or bypass mode, ensuring biocide-free operation.