Examination and chemical analysis of extremely well-preserved microfossils from the Lower Cambrian Lukati Formation in Estonia suggests that acritarchs from among the genera Globosphaeridium, Skiagia, Comasphaeridium and Lophosphaeridium have dinoflagellate affinities. The investigation presents a combination of transmitted light microscopy, fluorescence microscopy and flow cytometry, and biomarker analysis that demonstrates a new method for the investigation of problematic organic-walled microfossils. For the chemical analysis, Lukati Formation acritarchs were separated from prasinophycean tasmanitids by size and then divided into two fractions in accordance with the intensity of their autofluorescence signal. Biomarker molecules were generated by pyrolysis directly from isolated acritarch organic walls and studied using gas chromatography-mass spectrometry-mass spectrometry (GC-MS-MS) and metastable reaction monitoring (MRM)-GC-MS. The analysis supported previously made suggestions that acritarchs include microorganisms of different biological affinities. All acritarch fractions contain the common steranes (cholestane, 24-methylcholestane and 24-ethylcholestane) that are characteristic molecules for eukaryotes. However, the dinoflagellate-related biomarkers, dinosterane and 4α-methyl-24-ethylcholestane, were concentrated only in the fraction containing highly autoflourescent acritarchs. Additional chemical analyses of microfossils from the Lower Cambrian Buen Formation of North Greenland confirmed the presence of the dinoflagellate-related biomarkers at a second Early Cambrian locality.