Introduction Eliminating and/or inhibiting bacterial growth within the root canal system have been shown to play a key role in the regenerative outcome. The aim of this study was to synthesize and determine in vitro both the antimicrobial effectiveness and cytocompatibility of bi-mix antibiotic-containing polydioxanone (PDS)-based polymer scaffolds. Methods Antibiotic-containing (metronidazole, MET and ciprofloxacin, CIP) polymer solutions (distinct antibiotic weight ratios) were spun into fibers as a potential mimic to the double antibiotic paste (DAP, a MET/CIP mixture). Fiber morphology, chemical characteristics, and tensile strength were evaluated by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and tensile testing, respectively. Antimicrobial efficacy was tested over time (aliquot collection) against Enterococcus faecalis (Ef), Porphyromonas gingivalis (Pg), and Fusobacterium nucleatum (Fn). Similarly, cytotoxicity was evaluated in human dental pulp stem cells (hDPSCs). Data were statistically analyzed (p<0.05). Results SEM and FTIR confirmed that electrospinning was able to produce antibiotic-containing fibers with diameter mostly in the nanoscale. Tensile strength of 1:1MET/CIP scaffolds was significantly (p<0.05) higher than pure PDS (control). Meanwhile, all other groups presented similar strength as the control. Aliquots obtained from antibiotic-containing scaffolds inhibited growth of Ef, Pg and Fn, except pure MET that did not show an inhibitory action towards Pg or Fn. Antibiotic-containing aliquots promoted slight hDPSCs viability reduction, but none of them were considered to be cytotoxic. Conclusion Our data suggest that the incorporation of multiple antibiotics within a nanofibrous scaffold holds great potential towards the development of a drug delivery system for regenerative endodontics.