Laser interferometry is a measurement technique used in physical sciences, with a potential for new applications in microbiology. Our previously studies, focused on the quantitative analysis of antibiotics diffusion through membranes or their releasing from gel structure, indicate that this method might be useful in analysis of substances diffusion across the bacterial biofilms. As antibiotic - biofilm interaction model, we tested above method for determination of ciprofloxacin or gentamicin diffusion through Proteus mirabilis O18 biofilm. Laser interferometry analysis of antibiotics diffusion showed that the amount of ciprofloxacin transported through mature biofilm is 1.9 times higher than gentamicin. It was correlated with lower level of gentamicin in compare to the level of ciprofloxacin in biofilm, which amounts were predicted in biofilm during diffusion process by laser interferometry method. We suggest that the analysis of antibiotic diffusion in biofilm might by helpful in evaluation of effectiveness of antibacterial agents.