For the decontamination of surfaces in the food production industry, plasma generated compounds (PGCs) such as plasma-treated water (PTW) or plasma processed air (PPA) 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. 10 ml deionized water was treated with the MidiPLexc plasma source for 100 s, 300 s and 900 s (pre-treatment time) and the bacterial biofilms were exposed to the PTW for 1 min, 3 min and 5 min (post treatment time). To investigate the influence of PTW on P. fluorescens biofilms, microbiological assays (CFU, fluorescence and XTT assay) and imaging techniques (fluorescence microscopy, confocal laser scanning microscopy (CLSM), and atomic force microscopy (AFM)) were used. The CFU showed a maximum reduction of 6 log10 by using 300 s pre-treated PTW 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 was achieved by using 900 s pre-treated PTW for 5 min. The microscopic images showed evident microbial inactivation within the biofilm even at the shortest pre treatment (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. The findings correlated with a decrease in the pH value of the PTW, which forms the basis for the chemically active components of PTW and its antimicrobial effects. These results, provide valuable insights into the mechanisms of inactivation of biofilms by plasma generated compounds (PGCs) such as PTW and thus allow for further parameter adjustment for applications in food industry.