Honey bee propolis extract (HBP) was prepared by three methods; namely, soxhlet-reflux, magnetic stirring and ultrasound extraction. Based on the efficacies of HBP formation, the soxhlet-reflux method gave the highest yield of HBP. Electrospinning of cellulose acetate (CA) solution loaded with different concentrations of HBP was assessed. The results show that the surface morphology of CA nanofibers characterized by SEM changes with the addition of HBP to CA solution. The thermal stability of the as-formed CA/HBP marginally increases when compared with CA nanofibers. It was also observed that the CA/HBP nanofibers are effective inhibitors of growth of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Additionally, the in vitro release results of HBP from CA nanofibers displayed that the HBP release is sustained and controlled in neutral pH rather than alkaline and acid pH.