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With the emergence of antibiotic resistance, developing new antibiotics and therapies for combating bacterial infections is urgently needed. Herein, a series of quaternized fluorescent silicon nanoparticles (SiNPs) are facilely prepared by the covalent reaction between amine-functionalized SiNPs and carboxyl-containing N-alkyl betaines. It is found that the bactericidal efficacy of these quaternized SiNPs increases with the length of the N-alkyl chain, and SiNPs conjugated with N,N-dimethyl-N-octadecylbetaine (BS-18), abbreviated as SiNPs-C18, show the best antibacterial effect, whose minimum inhibitory concentrations for Gram-positive bacteria are 1–2 μg mL−1. In vivo tests further confirm that SiNPs-C18 have excellent antibacterial efficacy and greatly reduce bacterial load in the infectious sites. The SiNPs-C18 exhibit low cytotoxicity toward mammalian cells (including normal liver and lung cells, red blood cells, and macrophages), enabling them to be useful for clinical applications. Besides, the quaternized SiNPs exhibit polarity-dependent fluorescence emission property and can selectively image Gram-positive bacteria, thereby providing a simple method to successfully differentiate Gram-positive and Gram-negative bacteria. The present work represents the first example that successfully turns fluorescent SiNPs into metal-free NP-based antibiotics with simultaneous bacterial imaging and killing capability, which broadens the applications of fluorescent SiNPs and advances the development of novel antibacterial agents.