Retene and dehydroabietic acid methyl ester (DAME) are biomarkers linked mostly to terrigenous organic matter. However, their aquatic origin from algae and/or cyanobacteria has been discussed in the past few years. The complex origin of retene and DAME can lead to misinterpretation during biomarker interpretation, which can be minimalised with the support of isotopic analyses. This study contains the results of thirty-two compound-specific isotope analyses (CSIA) of δ13C performed on retene and forty-six CSIA analyses of δ13C performed on DAME. Obtained δ13C results were compared with simple biomarker results to tie isotopic data with the origin of organic matter. Pure aquatic retene and DAME were present within the Middle Cambrian and Early Ordovician samples, as the first appearance of plants was noted in the Middle Ordovician.CSIA was performed on a wide spectrum of the Cambrian-Silurian, Late Triassic-Middle Jurassic, and Neogene samples. Cambrian-Silurian strata include aquatic-origin retene and DAME, as evidenced by their comparatively low carbon isotopic signature. The average δ13C for retene and DAME within Cambrian-Silurian strata was equal to −31.7 ‰ and − 30.8 ‰, respectively. The Late Triassic-Middle Jurassic strata were dominated by terrigenous organic matter, resulting in higher δ13C values in comparison with the Cambrian-Silurian strata. The average δ13C for retene and DAME within the Late Triassic–Middle Jurassic strata was equal to −25.2 ‰ and − 26.3 ‰, respectively. The average δ13C for retene and DAME within the Neogene strata was equal to −27.3 ‰ in both cases, as these strata also contain a significant input of terrigenous organic matter. Some Neogene samples containing strong aquatic organic matter admixture are characterised by a lower δ13C for retene and/or DAME, oscillating around −28.0 ‰ to −30.0 ‰ for both compounds. Despite possible terrigenous input within some Silurian samples, the δ13C for retene and DAME still shows values typical for aquatic origin. These results suggest that retene and DAME within the Silurian strata should not be linked with non-vascular plants or that, due to their affinity with algae and cyanobacteria, the early plants were characterised by a relatively low δ13C composition, characteristic for phytoplankton organic matter. δ13C of retene and DAME were compared with δ13C of C16-C30n-alkanes and stigmastane, which supported the thesis about isotopic differences in aquatic and terrigenous retene and DAME.