The present study examined the biosynthesis and characterization of selenium nanoparticles (SeNPs) using two contrasting endophytic selenobacteria, one Gram-positive (Bacillus sp. E5 identified as Bacillus paranthracis) and one Gram-negative (Enterobacter sp. EC5.2 identified as Enterobacter ludwigi), for further use as biofortifying agents and/or for other biotechnological purposes. We demonstrated that, upon regulating culture conditions and selenite exposure time, both strains were suitable "cell factories" for producing SeNPs (B-SeNPs from B. paranthracis and E-SeNPs from E. ludwigii) with different properties. Briefly, dynamic light scattering (DLS), transmission electron microscopy (TEM), and atomic force microscopy (AFM) studies revealed that intracellular E-SeNPs (56.23 & PLUSMN; 4.85 nm) were smaller in diameter than B-SeNPs (83.44 & PLUSMN; 2.90 nm) and that both formulations were located in the surrounding medium or bound to the cell wall. AFM images indicated the absence of relevant variations in bacterial volume and shape and revealed the existence of layers of peptidoglycan surrounding the bacterial cell wall under the conditions of biosynthesis, particularly in the case of B. paranthracis. Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), energy-dispersive X-ray (EDS), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) showed that SeNPs were surrounded by the proteins, lipids, and polysaccharides of bacterial cells and that the numbers of the functional groups present in B-SeNPs were higher than in E-SeNPs. Thus, considering that these findings support the suitability of these two endophytic stains as potential biocatalysts to produce high-quality Se-based nanoparticles, our future efforts must be focused on the evaluation of their bioactivity, as well as on the determination of how the different features of each SeNP modulate their biological action and their stability.