The quorum sensing signals-acyl homoserine lactones (AHLs) were directly detected in 1-9-day-old subtidal biofilms developed in a coastal fish farm by using AHL reporter strains and gas chromatography-mass spectrometry. Both methods showed that the AHL molecules and/or AHL-producing bacterial community were dynamic during biofilm development, with dominant AHLs changed from short-chain to long-chain AHLs. Terminal restriction fragment length polymorphism analysis of the bacterial 16S rRNA genes derived from subtidal biofilms of different ages was compared to that of the 21 AHL-producing bacteria isolated from the same batch of subtidal biofilms. All terminal restriction fragments (TRFs) generated from AHL-producing bacteria matched with the dominant TRFs derived from the biofilm bacterial community samples. Particularly, the TRFs of all AHL-producing Vibrio spp. matched with the TRFs that were dominant only in 1-day-old biofilm, suggesting that AHL-producing vibrios were one of the pioneer groups during subtidal biofilm formation. We reported here for the first time the dynamics of AHLs and AHL-producing bacteria during the formation of a subtidal biofilm.