Fecal pollution affects environmental waters through spreading of animal manure and runoff of agricultural soils. However, the indicator bacteria (E. coli and enterococci) do not identify the source of the contamination and the development of fecal source tracking tools is crucial for water management and remediation. The aim of our study was to evaluate the transfer of chemical and microbial fecal markers during a runoff simulation on an agricultural plot previously spread with bovine or pig manure. Four types of markers have been analysed: 15 fecal sterols and stanols quantified by gas chromatography-mass spectrometry, chemical fingerprint obtained by 3D-fluorescence excitation-matrix spectroscopy, F-specific RNA specific bacteriophages (FRNAPH) and bacterial markers belonging to the Bacteroidales (Rum-2bac and Pig-2-Bac) and to the species Lactobacillus amylovorus. The results show that animal FRNAPH genogroups, sitostanol/coprostanol and cholestanol/cholesterol steroids ratios and tryptophan/ fulvic-like fluorescence differentiated pig and bovine fecal contamination. Sitostanol/coprostanol > 1 and cholestanol / cholesterol <1 are specific of bovine manure amended plot, whereas sitostanol / coprostanol <1 and cholestanol / cholesterol > 1 are characteristic of pig manure amended plot. L-amylovorus and Pig2-bac were only present in runoff water collected on pig manure amended plot whereas Rum2-bac was only detected in runoff water collected on bovine manure amended plot. Combined analyses of chemical and microbial markers provide relevant information on the determination of fecal contamination during runoff on agricultural soils. The next step is to test the applicability of these markers in watercourses submitted to nonpoint source fecal pollution.