A new online methodology based on a continuous process video microscopy and image analysis has been developed to study the effects of enzymes on the formation of biofilm. This research consists of two parts: (1) the monitoring of the growth of a biofilm formed with the axenic culture isolated from the process waters of a recycling paper mill, aiming at determining the most appropriate way to quantify the biofilm growth from the obtained images; and (2) the study of the effects of three new enzymatic products on biofilm formation involving the natural flora of microorganisms present in the process water of the paper mill. The results demonstrate that the new methodology based on image analysis allows monitoring of the formation of biofilm and selecting the most efficient biofilm control product. The study also shows that enzymatic products could be an alternative for biocides on biofilm prevention and control.