Conodont biogenic apatite has become a preferred analytical target for oxygen isotope studies investigating ocean temperature and palaeoclimate changes in the Palaeozoic. Despite the growing application in geochemically-based palaeoenvironmental reconstructions, the paucity or absence of conodont fossils in certain facies necessitates greater flexibility in selection of robust oxygen-bearing compounds for analysis. Vertebrate microfossils (teeth, dermal denticles, spines) offer a potential substitute for conodonts from the middle Palaeozoic. Vertebrate bioapatite is particularly advantageous given a fossil record extending to the present with representatives across freshwater to fully marine environments, thus widening the scope of oxygen isotope studies on bioapatite. However, significant tissue heterogeneity within vertebrates and differential susceptibility of these tissues to diagenetic alteration have been raised as potential problems affecting the reliability of the oxygen isotope ratios as palaeoclimatic proxies. Well-preserved vertebrate microfossils and co-occurring conodont fossils from the Upper Devonian and Lower Carboniferous of the Lennard Shelf, Canning Basin, Western Australia, were analysed using bulk (gas isotope ratio mass spectrometry, GIRMS) and in-situ (secondary ion mass spectrometry, SIMS) methodologies, with the latter technique allowing investigation of specific tissues within vertebrate elements. The δ18Oconodont results may be interpreted in terms of palaeolatitudinally and environmentally sensible palaeo-salinity and -temperature and provide a baseline standard for comparison against vertebrate microfossil δ18O values. Despite an absence of obvious diagenetic modification, GIRMS of vertebrate denticles yielded δ18O values depleted in 18O by 2–4‰ relative to co-occurring conodonts. SIMS analysis of dentine tissues exhibited significant heterogeneity, while hypermineralised tissues in both scales and teeth produced δ18O values comparable with those of associated conodonts. The susceptibility of permeable phosphatic fossil tissues to microbial activity, fluid interaction and introduction of mineral precipitates post-formation is demonstrated in the dentine of vertebrate microfossils, which showed significant heterogeneity and consistent depletion in 18O relative to conodonts. The hypermineralised tissues present in both teeth and scales appear resistant to many diagenetic processes and indicate potential for palaeoclimatic reconstructions and palaeoecological investigations.