Synthesis of biocompatible polymer nanofibers is valuable, due to their use as a cover for burns and as a replacement for bandage because of their antimicrobial properties. In this study, electrospinning of chitosan(Ch) and nanofibers synthesis with antibacterial properties was investigated. Nanofibers with antibacterial properties were synthesized by electrospun of Ch/poly(L-lactide)(PLA)/Imipenem(Imi) polymer solution. The results showed that the optimized ratio of Ch/PLA polymer solution was ratio of 50:50 and Ch 2 wt% and PLA 10 wt% polymer solution was the best weight percentage for nanofiber preparation. Also, the average diameter of Ch/PLA/Imi nanofibers was 143 nm and measured with ImageJ software. Afterwards, the antibacterial properties of Imi as additives (with different percentages) was studied in the polymer solution. The scanning electron microscopy (SEM) images and antibacterial tests were showed that the electrospun of Ch/PLA/Imi polymeric nanofibers were effective against Gram negative bacteria Escherichia coli (E. coli) and inhibited growth of E. coli. The growth and viability percentage of fibroblast cells with nanofibers in alpha MEM culture are at desirable levels after 6 days.