Microbial nanotechnology is an expanding research area devoted to producing biogenic metal and metalloid nanomaterials (NMs) using microorganisms. Often, biogenic NMs are explored as antimicrobial, anticancer, or antioxidant agents. Yet, most studies focus on their applications rather than the underlying mechanism of action or toxicity. Here, we evaluate the toxicity of our well-characterized biogenic selenium nanoparticles (bSeNPs) produced by the strain SeITE02 against the model yeast comparing it with chemogenic SeNPs (cSeNPs). Knowing from previous studies that the biogenic extract contained bSeNPs in an organic material (OM) and supported here by Fourier transform infrared spectroscopy, we removed and incubated it with cSeNPs (cSeNPs_OM) to assess its influence on the toxicity of these formulations. Specifically, we focused on the first stages of the eukaryotic cell exposure to these samples─i.e., their interaction with the cell lipid membrane, which was mimicked by preparing vesicles from yeast polar lipid extract or phosphatidylcholine lipids. Fluidity changes derived from biogenic and chemogenic samples revealed that the bSeNP extract mediated the overall rigidification of lipid vesicles, while cSeNPs showed negligible effects. The OM and cSeNPs_OM induced similar modifications to the bSeNP extract, reiterating the need to consider the OM influence on the physical-chemical and biological properties of bSeNP extracts.