Balanus amphitrite cyprids produce complex adhesive substances that enable their attachment to surfaces and impart a strong detachment resistance from most immersed substrata. The colonization of man‐made structures by barnacle cyprids and other marine organisms is a troublesome and costly phenomenon, for which controlling strategies are actively sought. In this work, we expand previous investigations about the susceptibility of cyprid adhesives to unpurified proteases in solution by evaluating the interplay between these secreted biomolecules and a surface‐confined purified protease. The strategy involved the covalent immobilization of the enzyme Subtilisin A to maleic anhydride copolymer thin films through the spontaneous reaction of anhydride moieties with lysine side chains. This enabled the production of bioactive layers of tunable enzyme surface concentration and activity, which were utilized to systematically evaluate the effect of the immobilized enzyme on cyprid settlement and exploratory behavior. Surfaces of increasing enzyme activity displayed a gradual decrease in cyprid settlement levels (approaching inhibition) as well as an increase in post‐settlement adhesion failure (evidenced by significant numbers of detached metamorphosed individuals). High activities of the bound enzyme also affected pre‐settlement behavior of cyprids, reducing the velocity and total distance moved while increasing the amount and speed of meander compared to the controls. The here‐reported low enzyme surface concentrations found to be remarkably effective at reducing cyprid settlement hold promise for the use of immobilized enzymes in the control of marine biofouling.