The conventional treatment of periodontal disease does not solve the high incidence of recolonization of periodontal pockets by pathogens. Here, we introduce an innovative concept of incorporating autochthonous bacteria as potential probiotics into nanofibers for local treatment. We selected and isolated the strain 25.2.M from the oral microbiota of healthy volunteers. It was identified as Bacillus sp. based on 16S rRNA sequence analyses. The strain is nonpathogenic, produces antimicrobial substances, and can grow over the periodontal pathogen Aggregatibacter actinomycetemcomitans in vitro, making it a promising probiotic candidate. The strain 25.2.M was successfully incorporated into the nanofibers in the form of spores (107 CFU/mg), the viabilities of which were exceptional (max. change of 1 log unit) both during electrospinning and after 12 months of storage. The release of the bacteria was delayed from chitosan/poly(ethylene oxide) compared to poly(ethylene oxide) nanofibers, and the antimicrobial activity against A. actinomycetemcomitans was confirmed. The developed nanodelivery system for administration into periodontal pockets thus offers a promising approach for the inhibition of periodontal pathogens and restoration of healthy oral microbiota.