Larval settlement of the Pacific oyster Crassostrea gigas on microbial biofilms obtained by immersing half-size glass slides in the sea (1.0 m depth) off Taira-cho, Nagasaki, Japan for 1 to 24 days during the period between May 2009 and January 2010 was investigated. Settlement inducing activities of biofilms that were subjected to heat (80°C), formalin (FA) and antibiotic mixture (AM) treatments were also investigated. Moreover, the settlement inducing activities of 4 bacterial strains isolated from the biofilm were investigated. C. gigas larvae settled in response to microbial biofilms. The percentage of post larvae increased with immersion period in biofilms obtained during Jan to Mar and Oct to Dec. Larval settlement also increased with the bacterial density of biofilms in Jan to Jun and Oct to Dec, and with the diatom density in Apr to Jun. By contrast, larval settlement did not linearly correlate with the dry weight of the biofilms. FA treatment did not affect the activity of the biofilm but heat and AM treatments of the biofilm resulted in significantly low percentage of post larvae. Of the 4 bacterial isolates tested, Pseudoalteromonas tetraodonis and Pseudoalteromonas sp. induced the highest percentage of post larvae but their activities were reduced with formalin treatment. Thus, microbial biofilms may possess a cue that remains intact even after killing the components of the film by FA treatment, and this cue may be distinct from the water soluble metabolite produced by specific bacterial species, such as Pseudoalteromonas sp.