Gold nanoparticles immobilized on gold surfaces (AuNPs/Au) function as an excellent SERS substrate for the detection of bacteria biomarkers. The possibility of the reactivity of bacteria biomarkers on such a nanoparticle-based substrate poses complications for the spectroscopic identification and quantification. This report describes new findings of an investigation of the SERS characteristics for the competitive adsorption of dipicolinic acid (DPA), which is an important biomarker from bacterial spores, and pyridine (Py), which is a possible decarboxylation product of DPA. The comparison focuses on the diagnostic region of 900–1100 cm⁻¹ associated with the ring-breathing modes of the two molecules. While the SERS spectra in this region appeared to display some similarities between DPA and Py, distinctive differences in the detailed band characteristics were revealed for both individual and competitive adsorptions on the AuNPs/Au substrates. The fact that the equilibrium constant for the adsorption of Py on the substrate (∼8 × 10⁵ M⁻¹) is larger than that for DPA (∼2 × 10⁵ M⁻¹) in the measured concentration region is attributed to a stronger binding of Py to Au surface than that for DPA. The analysis of the differences has provided not only accurate speciation of the biomarker molecules on the gold nanoparticle based substrates under the SERS measurement conditions, but also has implications for expanding the application of the nanoparticle substrates for highly sensitive and selective detection of bacterial biomarkers under various reactive or non-reactive conditions.