ABSTRACT Microbiota, the microorganisms that colonize living organisms. Oysters rely exclusively on an innate immune system. This study characterized the cultivable gastrointestinal tract microbiota (GTM) of the oyster Crassostrea gasar and evaluated their influence on immune responses. Antibiotics were used to reduce or alter the microbiota during in vitro and in vivo assays. Haemocyte viability, concentration, and phagocytic capacity, as well as the production of reactive oxygen species (ROS), were evaluated in antibiotic-treated and control oysters. Microbiological analysis of the gastrointestinal tract was also performed; bacteria were selected using culture media and were identified by 16S ribosomal DNA. The results showed that the antibiotics eliminated bacteria in vitro but increased their concentrations in vivo. The GTM was composed of 26% amylolytic bacteria, 21% lipolytic bacteria, 18% proteolytic bacteria, 18% cellulolytic bacteria and 17% lactic acid bacteria; there were no differences in the amounts of these bacterial types between the control and treated oysters. Three major bacterial phyla, Proteobacteria, Firmicutes and Actinobacteria, and seven genera, Labrenzia, Pseudomonas, Halomonas, Shewanella, Vibrio, Bacillus, and Micrococcus, were detected. The concentration, viability, and phagocytic capacity of hemocytes and the production of ROS were not significantly altered by antibiotic treatment. In conclusion, the antibiotics altered the number of heterotrophic bacteria without changing hemocyte function, suggesting that this assay could be useful for verifying the influence of microbiota in host-parasite interactions.