Indole derivatives derived from the secondary metabolites of marine organisms possess the excellent antifouling property to inhibit the biofouling. These compounds and their analogues are simple in structure and have been proven to have low toxicity and bioaccumulation. Therefore, the active indole antifoulants are expected to replace the potentially toxic antifoulants which are widely used in current antifouling coatings. Seven indole derivatives were synthesized via the Friedel-Crafts alkylation reaction and were characterized by IR spectra, 1H NMR, 13C NMR and elemental analysis. Inhibition experiments against marine algae and bacteria were conducted, and the partial inhibition rates of algae and bacteria were more than 90%. This outcome indicates that indole derivatives possess excellent properties suitable for use as targeting anti-fouling compound for algae and bacteria. Non-invasive Micro-test Technology (NMT) reveals that the Ca2+ efflux of Platymonas subcordiformis dramatically increased in the presence of indole derivatives, which is inferred to be the molecular mechanism for inhibiting the growth of marine algae. The antifouling coatings containing indole derivatives were prepared and subjected to an antifouling test in a marine environment, and the results show that N-(1-H-5-bromo-indole-3-ylmethyl) benzamide and N-(1-H-2-phenyl-indole-3-ylmethyl) benzamide possess better antifouling performance compared to copper pyrithione (CuPT). According to these results, indole derivatives in this study might become novel and promising antifoulants.