Fluorescence excitation-emission matrices (FEEMs) and parallel factor analysis (PARAFAC) techniques are able to evaluate the chemical characteristics of overlapping fluorophores and bands in FEEM. In this study, FEEM-PARAFAC was applied to distinguish the differences among metabolic organic components from Pseudomonas fluorescents P17 and Spirillum NOX strains in an assimilable organic carbon bioassay with acetate as the primary substrate. Three metabolic components from P17 and NOX strains were selected and grown in different acetate concentrations based on the simultaneous consideration of verification of variance, core consistency, residuals, and split-half experiments. The results from control tests show that metabolic humic-like substances in the NOX strain were more prevalent than they were in the P17 strain. Metabolic humic-like substances from the P17 strain increased with escalating acetate concentrations, while a descending trend occurred in experiments with the NOX strain, indicating that the composition of metabolic organic matter containing nitrogen and humic-like substances varied according to the acetate concentrations.