For the decontamination of surfaces in the food production industry, plasma-generated compounds such as plasma-treated water or plasma-processed air offer many promising possibilities for future applications. Therefore, the antimicrobial effect of water treated with microwave-induced plasma (MidiPLexc) on Pseudomonas fluorescens biofilms was investigated. A total of 10 mL deionized water was treated with the MidiPLexc plasma source for 100, 300 and 900 s (pretreatment time) and the bacterial biofilms were exposed to the plasma-treated water for 1, 3 and 5 min (post-treatment time). To investigate the influence of plasma-treated water on P. fluorescens biofilms, microbiological assays (colony-forming units, fluorescence and XTT assay) and imaging techniques (fluorescence microscopy, confocal laser scanning microscopy, and atomic force microscopy) were used. The colony-forming units showed a maximum reduction of 6 log10 by using 300 s pretreated plasma water for 5 min. Additionally, a maximum reduction of 81% for the viability of the cells and a 92% reduction in the metabolic activity of the cells were achieved by using 900 s pretreated plasma water for 5 min. The microscopic images showed evident microbial inactivation within the biofilm even at the shortest pretreatment (100 s) and post-treatment (1 min) times. Moreover, reduction of the biofilm thickness and increased cluster formation within the biofilm was detected. Morphologically, the fusion of cell walls into a uniform dense cell mass was detectable. The findings correlated with a decrease in the pH value of the plasma-treated water, which forms the basis for the chemically active components of plasma-treated water and its antimicrobial effects. These results provide valuable insights into the mechanisms of inactivation of biofilms by plasma-generated compounds such as plasma-treated water and thus allow for further parameter adjustment for applications in food industry.