Positively charged C-dot/Ag composite nanoparticles were synthesized via the facile one-step hydrothermal reaction of L-arginine and silver nitrate. L-arginine was used not only as the carbon and nitrogen sources of N-doped C-dots but also as the reducing agent of silver ions. It was noteworthy that the resulting C-dots were negatively charged but the simultaneous reduction of silver ions made the resulting C-dot/Ag composite nanoparticles become positively charged. Furthermore, as compared to C-dots, the presence of Ag nanoparticles and the higher nitrogen content led to the redshift of excitation and emission intervals. Also, the enlarged excitation wavelength range in the visible light region made the resulting C-dot/Ag nanocomposite more useful in fluorescence imaging. In addition, the C-dot/Ag composite nanoparticles exhibited more excellent bacteria-killing capability than C-dots and were successfully used for the fluorescence imaging of E. coli because they could attach and release silver ions on the surface of E. coli. In conclusion, a facile one-step hydrothermal process has been successfully developed for the synthesis of C-dot/Ag composite nanoparticles, and the resulting C-dot/Ag composite nanoparticles are expected to have great potential in the killing and fluorescence imaging of Gram-negative bacteria.