Biofilm formation results in medical threats or economic losses and is therefore a major concern in a variety of domains. In two-species biofilms of marine bacteria grown under dynamic conditions, Pseudoalteromonas sp. strain 3J6 formed mixed biofilms with Bacillus sp. strain 4J6 but was largely predominant over Paracoccus sp. strain 4M6 and Vibrio sp. strain D01. The supernatant of Pseudoalteromonas sp. 3J6 liquid culture (SN(3J6)) was devoid of antibacterial activity against free-living Paracoccus sp. 4M6 and Vibrio sp. D01 cells, but it impaired their ability to grow as single-species biofilms and led to higher percentages of nonviable cells in 48-h biofilms. Antibiofilm molecules of SN(3J6) were able to coat the glass surfaces used to grow biofilms and reduced bacterial attachment about 2-fold, which might partly explain the biofilm formation defect but not the loss of cell viability. SN(3J6) had a wide spectrum of activity since it affected all Gram-negative marine strains tested except other Pseudoalteromonas strains. Biofilm biovolumes of the sensitive strains were reduced 3- to 530-fold, and the percentages of nonviable cells were increased 3- to 225-fold. Interestingly, SN(3J6) also impaired biofilm formation by three strains belonging to the human-pathogenic species Pseudomonas aeruginosa, Salmonella enterica, and Escherichia coli. Such an antibiofilm activity is original and opens up a variety of applications for Pseudoalteromonas sp. 3J6 and/or its active exoproducts in biofilm prevention strategies.