We have developed a one-step method to synthesize carbon quantum dots (CQDPAs) from biogenic polyamines (PAs) as an antibacterial agent for topical treatment of bacterial keratitis (BK). CQDs synthesized by direct pyrolysis of spermidine (Spd) powder through a simple dry heating treatment exhibit a solubility and yield much higher than those from putrescine and spermine. We demonstrate that CQDs obtained from Spds (CQDSpds) possess effective antibacterial activities against non-multidrug-resistant Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella enterica serovar Enteritidis bacteria and also against the multidrug-resistant bacteria, methicillin-resistant S. aureus. The minimal inhibitory concentration (MIC) of CQDSpds is ∼2500-fold lower than that of spermidine alone, demonstrating their strong antibacterial capabilities. Investigation of the possible mechanisms behind the antibacterial activities of the as-synthesized CQDSpds indicates that the super-cationic CQDSpds with small size (diameter ca. 6 nm) and highly positive charge (ζ-potential ca. +45 mV) cause severe disruption of the bacterial membrane. In vitro cytotoxicity, hemolysis, hemagglutination, genotoxicity, and oxidative stress and in vivo morphologic and physiologic cornea change evaluations show the good biocompatibility of CQDSpds. Furthermore, topical ocular administration of CQDSpds can induce the opening of the tight junction of corneal epithelial cells, thereby leading to great antibacterial treatment of S. aureus-induced BK in rabbits. Our results suggest that CQDSpds are a promising antibacterial candidate for clinical applications in treating eye-related bacterial infections and even persistent bacteria-induced infections.