Biofilms are groups of microorganisms which are attached to the surface or which occur at different interphases. They represent a big problem because they are difficult to remove, and they cause significant costs in the industry if they occur in the pipework. In this thesis, we focused on the impact of the environment on the formation of biofilms and the effect of their structure on the effectiveness of antimicrobial agents. We used different types of surfaces and nutrients (different carbon sources and minimum/rich media) in which we have grown biofilms. We used microscopy and rheology to determine viscosity and elasticity of resulting biofilms. We treated the most structurally different biofilms with various non-commercial and commercial antimicrobials and compared the percentage of dead cells in the biofilm after treatment. While growing biofilms, we have established the conditions which enabled the formation of the vertical gradient of the biofilm covered surface. Biofilm covered surface was dependent on the type of surface and carbon source. We saw that agents that we used didn’t completely destroy all the cells in the biofilm. The effectiveness of antimicrobials was decreasing whenever the biofilm surface coverage was increased. It seems that the effectiveness of biocides depends on the rheological properties of the biofilm. Similarly to the biofilm, we observed that in planktonic form there is some kind of anticorrelation between the density of the bacterial culture and the effectiveness of the biocide.