Mustard seed containing glucosinolates can be used as a natural source of antimicrobial volatile isothiocyanate (ITC) through enzymatic hydrolysis. ITC can be used in active food packaging to inhibit the proliferation of spoilage microorganisms. In this study, antibacterial composite fibers were prepared by encapsulating mustard powder (MP) in carrier materials of electrospun poly(L-lactic acid) (PLLA)/poly[(lacticacid)-co-lysine] (PLAL) fibers. Uniaxial composite fibers (PLLA/MP, PLLA/PLAL/MP) and coaxial composite fibers (PLLA/PLAL/MP-PLLA, PLLA/MP-PLLA/PLAL) were fabricated by electrospinning, and these fibers could be used to produce a series of volatile antibacterial substances, ITCs, which were extracted by hexane under proper temperature and moisture. The morphologies, chemical structures, thermal properties and hydrophilicities of the electrospun composite fibers were investigated. Ultraviolet spectrophotometry (UV) was used to evaluate the controlled ITC release behaviors from these fibers. The results indicated that the mean particle diameter was 975 +/- 126 nm and the main interaction between the MP and the carrier polymers was physical. The addition of MP and PLAL could improve the hydrophilic property of PLLA and change the thermal properties. Different fibers exhibited distinct ITC release behaviors, and the ITC release period of the coaxial fibers (approximately 14 days) was longer than that of the uniaxial fibers (approximately 8 days). These fibers have bright prospects in antibacterial ITC controlled release.