This study introduces novel chitosan (CS) and polyethylene oxide (PEO) copolymers reinforced with bacterial cellulose (BC) to fabricate nanofibers using the electrospinning method. SEM analysis confirmed uniform nanofiber formation, with CS/PEO/BC nanofibers (~240 nm) exhibiting a larger diameter than CS/PEO ones (~190 nm). FTIR spectroscopy confirmed BC integration, while Differential scanning calorimetry analysis indicated minimal impact on glass transition temperature. Notably, as compared to CS/PEO nanofibers, the CS/PEO/BC ones demonstrated superior swelling capacity, accelerated biodegradation, and enhanced mechanical (i.e., tensile) properties, with maximum stress and strain values of ~3.41 MPa and ~0.01% vs. ~2.14 MPa and ~0.01%. Antimicrobial assays confirmed activity against bacterial strains, and biocompatibility tests showed high cell viability at day seven (99.26% for CS/PEO/BC nanofibers). These findings highlight the potential of CS/PEO/BC nanofibers as promising candidates for tissue engineering, offering improved strength, biodegradability, and antimicrobial properties.