From an environmental perspective, there is a need to reduce the amount of biocides in chemically-active antifouling (AF) coatings, as these biocides can cause severe damage to marine life. In this study, six active molecules known as booster biocides were added at 3 wt% in solvent and aqueous-based acrylic paints. DCOIT, dichlofluanid, zinc pyrithione, copper pyrithione, tralopyril and zineb were used alone and mixed with a lower amount of copper (I) oxide and copper thiocyanate (10 wt%) than is currently used or with zinc oxide fillers. Field immersion tests were carried out in the Atlantic Ocean (Ste Anne du Portzic) and the Mediterranean Sea (Toulon), with contrasting physicochemical properties of water and macrofoulers' biodiversity. Microscopic analyses showed that AF coatings reduced the bacterial density differently for short immersion times at Ste Anne du Portzic. After four/five months of immersion, results, including multidimensional analyses, showed first specific patterns in the macrofoulers' assemblages depending on the site. In addition, the structure of macrofoulers' assemblages was affected by the biocide itself and by the combination of the biocide and the binder. When efficient, AF coatings appeared to decrease the richness with a strongest effect at Toulon. DCOIT was the most performing booster biocides whatever the immersion site and the polymer binder used. DCOIT was found to be efficient against marine bacteria and macrofoulers at Ste Anne du Portzic. The combination of zinc pyrithione with ZnO fillers in an aqueous-based AF paints exhibited the highest activity in the Mediterranean Sea over 16 months of immersion.