Biofilms are dynamic structures constituted by microorganisms that grow and die, and understanding these processes may be crucial to control biofilm development in various environments. Assuming a generally accepted first order decay kinetics of biofilm mass in time, mean residence time can be calculated. Using the initial labeling of the biofilm by ¹³C stable isotope, we were able to determine the residence time of the carbon in physiologically active biofilms. Our data indicate that the residence time is strongly affected by nutrition and differs substantially between biofilms formed by different bacterial isolates. Moreover, the biofilm formed from mixed soil inocula showed almost the same carbon residence time as the biofilms formed from both soil inocula applied separately. This does not indicate the existence of dramatic incompatibility between members of two interacting microbial communities. In the situation when the established biofilm biomass undergoes continuous replacement by newly appearing cells, the complex biofilm admit reluctantly the newly arriving microorganisms as components of the existing community. Our study represents a new insight into the biofilm dynamics in vitro.