The authors present bio-optical data spanning 316 sets of observations made at 34 inland waterbodies in Australia. The data was collected over the period 2013–2021 and comprise radiometric measurements of remote sensing reflectance (Rrs), diffuse attenuation extinction coefficient (Kd); optical backscattering; absorption of coloured dissolved organic matter (aCDOM), phytoplankton (aph) and non-algal particles (aNAP); HPLC analysis of algal pigments including chlorophyll-a (CHL-a); organic and inorganic total suspended solids (TSS); and total and dissolved organic carbon concentration. Data collection has been timed to coincide with either Landsat 8 or Sentinel-2 overpasses. The dataset covers a diverse range of optical water types and is suitable for algorithm development, satellite calibration and validation as well as machine learning applications.