To help understand bioapatite microstructures and related chemical variations, their impact on O‐isotope compositions measured and give insights on sample preparation, this study analysed conodonts and shark teeth prepared in different orientations through microanalytical and bulk sampling techniques: scanning electron microscopy (SEM); electron probe microanalysis (EPMA); continuous‐flow and high‐temperature reduction – isotope ratio mass spectrometry; and secondary ion mass spectrometry (SIMS). The SEM and EPMA measurements in conodonts allowed to distinguish the tissues commonly analysed by SIMS, which included albid and hyaline crowns but given their often small‐scale intergrowth, mixtures of these are difficult to avoid. In situ SIMS O‐isotope analyses provided different δ18O values: lower values with higher variance (16 ± 1‰ n = 13, 15.7 ± 1.9‰ n = 11) for mixed albid‐hyaline tissues, and higher, homogeneous values (17.1 ± 0.2‰, n = 13) for mainly hyaline tissues. Recent shark teeth δ18OSIMS value for dentine of the same tooth was 10‰ lower than the mean δ18OSIMS value for enameloid whereas the δ18OPO4 values measured for enameloid and dentine using the HTR method were identical. The variation of δ18O seems sensitive to analytical artefacts related to sample textures, caused during the sample preparation over more porous biomineral surfaces.