Aims:Biofouling is a common biology phenomenon occuring on ship surface. This phenomenon has become serious threat in marine industries because of great economic loss. Tributyltin has been used to prevent biofouling, but it turned to cause the environmental problem. Therefore, the discovery of alternative environment-friendly compound is necessarily needed. Methodology and results: Five Actinobacteria isolates and fourteen marine bacteria isolates were tested against the biofilm formation of eight biofouling bacteria isolates that isolated from boat surface and the attachment of three biofouling diatoms (Amphora, Navicula, Nitzschia). Actinobacteria CW17 supernatant showed the broad spectrum activity against all fouling bacteria, whereas BC 11-5 supernatant was the only marine bacteria that capable to inhibit biofilm formation of V. neocaledonicus. Moreover, three representative diatoms attachment could be inhibited by the bioactive compounds produced by Actinobacteria and marine bacteria. CW01 supernatant showed broad spectrum and high activity against all three representative diatoms which is very promising. Molecular identification based on 16S rDNA gene sequence showed eight fouling bacteria isolates were biofilm-forming bacteria. Conclusions, significance and impact of study: This research showed aquatic Actinobacteria and coral-associated marine bacteria have the potential to prevent biofouling formation. Further studies are needed to purify and characterize these antibiofouling compounds for environmental application.