Aims This study evaluated methods to sample and extract nucleic acids from Pacific oysters to accurately determine the microbiome associated with different tissues. Methods and Results Samples were collected from haemolymph, gill, gut and adductor muscle, using swabs and homogenates of solid tissues. Nucleic acids were extracted from fresh and frozen samples using three different commercial kits. The bacterial DNA yield varied between methods (P < 0.05) and each tissue harboured a unique microbiota, except for gill and muscle. Higher bacterial DNA yields were obtained by swabbing compared to tissue homogenates and from fresh tissues compared to frozen tissues, without impacting the bacterial community composition estimated by 16S rRNA gene (V1-V3 region) sequencing. Despite the higher bacterial DNA yields with QIAamp (R) DNA Microbiome Kit, the E.Z.N.A.(R) Mollusc DNA Kit identified twice as many operational taxonomic units (OTUs) and eliminated PCR inhibition from gut tissues. Conclusions Sampling and nucleic acid purification substantially affected the quantity and diversity of bacteria identified in Pacific oyster microbiome studies and a fit-for-purpose strategy is recommended. Significance and Impact of the Study Accurate identification of Pacific oyster microbial diversity is instrumental for understanding the polymicrobial aetiology of Pacific oyster mortality diseases which greatly impact oyster production.