Aims: This study aimed to investigate the efficacy of neutral electrolysed water (NEW) on biofilm and planktonic forms of Listeria monocytogenes, Escherichia coli and Pseudomonas aeruginosa in Luria-Bertani (LB) broth, 0.5 and 3% w/w fat pasteurized and ultra high temperature (UHT) milk. Methods and Results: Biofilm was established on stainless steels in a simulated industrial model biofilm reactor. NEW had a bactericidal effect on planktonic forms of all bacteria at 25 mu g ml(-1) concentration. Biofilm production index (BPI) was affected by the type of media and micro-organisms and incubation temperatures. In general, biofilms were more intense at 22 degrees C in milk containing 3% fat than 0.5% fat and LB. The highest BPI (1.12) was reported for the biofilm of E.coli at 22 degrees C in 3% fat pasteurized milk. No significant change in biofilm formation for any bacteria at UHT and pasteurized milk was obtained. NEW at 75 mu g ml(-1) concentration had more significant biofilm removal activity on all the tested bacteria. The biofilm removal property of NEW in high-fat milk was lower than that in low-fat milk and LB broth. Conclusions: NEW can be used at 75 mu g ml(-1) concentration as a sanitizing and biofilm removal compound in dairy plant facilities. Significance and Impact of the StudyThis study highlighted the effect of different growth media, including culture media and different types of milk, and the biofilm removal activity of NEW on foodborne pathogens and spoilage bacteria which could be considered in plant sanitation schedule.