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Antibacterial composite nanofibers have recently been widely applied in biomedical fields. The purpose of this study is to combine Polyamide66 (PA) with carbon nanotubes (CNTs) and Ag nanoparticles through electrospinning and the aqueous reduction method to synthesis Ag@CNT/PA composite nanofibers with excellent conductivity, antibacterial property and cytocompatibility. The morphology and structure of Ag@CNT/PA composite nanofibers were analyzed by a series of characterizations. Conductivity of Ag@CNT/PA composite nanofibers in wet state was measured using a four-probe resistance tester. The antibacterial activity of Ag@CNT/PA composite nanofibers was tested by inhibition zone method, and the MG-63 cells were used to detect the cytotoxicological effects of the composite nanofibers. The results show that the Ag nanoparticles (50–100 nm) are distributed uniformly on the surface of nanofibers. Conductivity of Ag@CNT/PA composite nanofibers reaches (9.918 ± 0.043) × 10−4 S mm−1, significantly higher than that of PA nanofibers ((1.486 ± 0.017) × 10−4 S mm−1). The Ag@CNT/PA composite fibers present good antibacterial activity against Escherichia coli and Staphylococcus aureus. Cell culture results show that the cell proliferation of Ag@CNT/PA composite nanofiber group seems no significant difference with PA nanofiber group (p > 0.05) at day 7. The Ag@CNT/PA composite nanofibers have no significant negative effects on MG-63 cells. [ABSTRACT FROM AUTHOR]