The Influence of water source on the production of slightly acidic electrolyzed water (SAEW) and its sanitization efficacy were investigated. Two different water sources (tap water (TW) and underground water (UGW)) were applied to produce slightly acidic electrolyzed water (SAEW) at same setting current, with similar electrolyte flow rate (EFR) and concentration. Properties of SAEW were evaluated based on pH, Available chlorine concentration (ACC) and oxidation-reduction potential (ORP). Methods for the optimization of SAEW production process was examined to obtain high ACC value by implanting different types of electrolytes. Effect of ACC and pH of SAEW were evaluated in vitro towards inactivate foodborne pathogens. The results indicated that TW with hardness of 29 ppm produced effectively SAEW than through UGW (12 ppm) using electrolytes. Likewise, low water hardness could be reinforced by combining HCL with a salt (NaCl or KCL). The optimized SAEW production system was determined at 4% HCl + 2.0 M KCL with EFR of 2 mL/min and 4% HCl + 3.0 M KCL with EFR of 2 mL/min resulting in higher ACC value of 56.5 and 65.5 ppm, respectively using TW. Pathogenic vegetative cells were completely inactivated within 1 min of treatment in SAEW with 20 ppm. Viability observations using Confocal and TEM Microscopy, Flow cytometry, and antimicrobial activity were carried out to confirm the sanitizing effect and cell membrane disruption. Based on the experimental results obtained, it provides a foundation for future advancement towards commercial application of SAEW in the food and agricultural industries.