A membrane perfusion method for determining the minimum effective release rate (MERR) of antifouling agents is described. With this method precisely controlled and reproducible release rates can be achieved independent of water chemistry and flow near the immersed surface. Like immersed painted surfaces, it allows for the establishment of diffusion gradients so that fouling organisms may exhibit chemotactic responses to the released toxin. The method is applied to natural water microbial biofilms dominated by the protozoansZoothamniumspp. andCharchesiumspp. The MERR of TBTC1 for 90% fouling reduction was found to be 2–4 μg·cm−2d−1in the local estuary. This rate result is consistent with observations of antifouling paints in service, which have shown that biofilms can be present on TBT coatings which prevent barnacle and tubeworm fouling. Thus, alternative environmentally acceptable methods are needed if control of microbial biofilms, or slimes, is required.