Electrospun nanofibers have emerged as a promising platform for probiotic delivery, with bacterial preservation posing a significant challenge in formulation design. This study examined the preservation of bacteria in various poly(ethylene oxide)-based nanofiber formulations and the solid-state behaviour of the excipients after electrospinning and during 24 weeks of storage under different conditions. Nanofiber formulations were loaded with bacteria from three different genera (oral cavity isolates Staphylococcus 26.3.J and Stenotrophomonas 27.3.S and vaginal Lactobacillus jensenii) and supplemented with 5 different stabilizers (sucrose, trehalose, glucose, mannitol or dextran), some of which also demonstrated nutrient characteristics. Efficacy of the tested stabilizers was species-dependent, with dextran as the most effective stabilizer for Staphylococcus 26.3.J and Stenotrophomonas 27.3.S and sucrose for L. jensenii. Low molecular weight stabilizers underwent complete (trehalose) or partial (sucrose, glucose, mannitol) amorphization during electrospinning in most formulations. Proportions of amorphous fractions of the semi-crystalline stabilizers were significantly influenced by the bacterial species, reaching up to 36% for sucrose and 28% for mannitol. Over 24 weeks, trehalose remained fully amorphous, while semi-crystalline stabilizers demonstrated instability of amorphous fractions, which underwent crystallisation. Notably, for oral isolate probiotics, amorphous stabilizers trehalose and dextran outperformed almost all semi-crystalline alternatives in preserving bacterial viability. In contrast, mannitol and glucose occasionally even reduced survival compared to PEO-only formulations, pointing out potential risks associated with physical instability of excipients. This study highlights the importance of selecting stabilizers tailored to specific bacterial species and understanding the solid-state properties of excipients to enhance probiotic survival in nanofiber-based formulations.