In this study, we employed electrospinning to fabricate polylactic acid (PLA)/carbon nanotubes (CNTs)/chitosan (CS) composite fibers containing different CS contents and examined the ability of these fibers for strawberry preservation for the first time. The experimental results showed that as CS content increased, the mechanical properties, solubility, and swelling ratio of the fiber films first improved and then degraded, with peak values being achieved at a CS content of 7 wt%. The antimicrobial activity test results showed that as CS content increased, the antimicrobial activity of the composite fiber films against four microorganisms increased and the activity was the best when the CS content was 7 wt%. In addition, the composite fibers exhibited better antimicrobial activity against Gram-negative and -positive bacteria than against molds. Moreover, the fibers exhibited better antimicrobial activity against Staphylococcus aureus than against Escherichia coli. The preservation experiments showed that the nanocomposite fibers containing different CS contents exhibited good preservation effects for strawberries, with the fiber containing 7 wt% CS showing the best results. Thus, these fibers can delay the physiological changes in strawberries and extend their shelf life and therefore have important potential applications for fruit and vegetable preservation in the future.