Biogenic metal/metalloid nanoparticles of microbial origin retain a functional biomolecular capping layer that confers structural stability. Little is known about the composition of such capping material. In this study, selenium nanoparticles (SeNPs) synthesized by five different bacterial strains underwent comparative analysis with newly proposed protocols for quantifying the concentration of carbohydrates, proteins and lipids present in capping layers. SeNPs were therefore treated with two different detergents to remove portions of the surrounding caps in order to assess the resulting effects. Capping material quantification was carried out along with the measure of parameters such as hydrodynamic diameter, polydispersity and surface charge. SeNPs from the five strains showed differences in their distinct biomolecule ratios. On the other hand, structural changes in the nanoparticles induced by detergents did not correlate with the amounts of capping matrix removed. Thus, the present investigation suggests a hypothesis to describe capping layer composition of the bacterial SeNPs: some biomolecules are bound more strongly than others to the core metalloid matrix, so that the diverse capping layer components differentially contribute to the overall structural characteristics of the nanoparticles. Furthermore, the application of the approach here in combining quantification of cap-associated biomolecules with the measurement of structural integrity-related parameters can give the biogenic nanomaterial field useful information to construct a data bank on biogenically synthesized nanostructures.