We investigated the composition of dissolved organic matter (DOM) in the hypersaline Mono Lake in California (USA), providing a detailed molecular characterization of DOM in this extreme ecosystem during a shift in limnological conditions (2017–2018). This dataset contains DOM molecular data (mass-to-charge ratios and peak intensities) obtained using ultrahigh-resolution mass spectrometry (21T Fourier transform ion cyclotron resonance mass spectrometry, FT-ICR MS; Bruker). Water samples were collected seasonally at depths ranging from 0.5 m to 35 m, using a CTD rosette as described in Phillips et al. (2021). Additionally, the dataset includes DOM molecular data from two main creeks - Lee Vining Creek and Rush Creek - as well as from a dominant phytoplankton species, Picocystis sp.