During the AO2023-1 expedition (1-21 June 2023), Melosira assemblages were collected using a handpump at a sampling site in Fram Strait. In addition to performing physiological measurements and filtering the material for trophic markers (fatty acids, stable isotope ratios of fatty acids, sterols and highly branched isoprenoids (HBIs), living material was brought back to Akvaplan-niva in Oslo and two strains were established as unialgal cultures. The cultures are maintained in artificial seawater-based L1 medium at 4°C and 25 µmol m-2 s-1 light with a 16:8 L:D cycle. Lipids were extracted from freeze-dried samples following the method of Folch et al. (1957), using a dichloromethane/methanol mixture (2:1, v/v) in a sonication bath. Thereafter, lipids were saponified with 20% potassium hydroxide in water/methanol (1:9). Sterols were extracted with hexane, purified by open-column chromatography with SiO2 and eluted with methanol. Sterol fractions were derivatized using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). Sterols were analyzed with gas chromatography–mass spectrometry (Agilent 7890B, Agilent Technologies), equipped with a DB-1ms column (30 m length, 0.25 mm internal diameter, 0.25 μm film thickness) and an Agilent 5977A MSD, following Stein et al. (2025). The temperature program ranged from 60°C to 320°C. Sterols were quantified using an internal standard (androstanol). Sterol analysis was conducted to complement datasets on fatty acid proportions and fatty acid stable isotope compositions. Collectively, the datasets enable a detailed characterization of the biochemical composition of different Melosira arctica-dominated algal assemblages from the Arctic Ocean, and a comparison with a unialgal culture of this important species.