The Northern Patagonia coast, characterized by an intricate interaction among terrestrial and marine systems such as Reloncaví Estuarine System (RES), present highly productive marine and aquaculture activities, having a significant socio-economic importance in Chile. Understanding the composition of Organic Matter (OM) in aquatic ecosystems is crucial for elucidating biogeochemical processes, and the use of lipid biomarkers, has proven valuable in identifying OM sources. This study investigates the relationship between phytoplankton biomass indicators, including phytoplankton abundance, chlorophyll-a, and sterol molecules synthesized in high percentages by phytoplankton cells, also known as phytoplankton-derived sterols at the RES. The RES encompasses the Reloncaví Fjord (RF) and the Reloncaví Sound (RS) and exhibits a high influenced by oceanic waters and freshwater discharge from rivers Spatio-temporal sampling was conducted during the austral spring, summer and winter expeditions of 2018 and 2019, at two sites in RS and RF mouth (RFm). Our findings reveal higher sterol concentration at RFm than RS. Notably, high sterol concentrations during austral summer season coinciding with increased phytoplankton abundances. Furthermore, higher concentrations of terrestrial-derived sterols such as β-sitosterol (C29Δ5) and stigmasterol (C29Δ5,22), at RFm site suggested an input of OM from the inner RF. Phytoplankton-derived sterols such as 24-methylenecholesterol (C28Δ5,24(28)) provide a reliable estimate of total diatom abundance at both sites (RFm and RS), though they showed a lower relationship with taxonomic subroups and phytoplankton classes in our study area. Hence, phytoplankton-derived sterols can be considered reliable diatom biomarkers, particularly in the RS, where the primary source of OM is predominantly marine, and even with high sterol degradation values at RES. Our study highlights the importance of interpreting changes in sterol abundances as phytoplankton community shifts. To avoid misinterpretation, it is essential to incorporate direct phytoplankton counts in elucidating the complex biosynthetic sources of sterols within the water column.