Inability of antibiotics repertoire to effectively control the progress of multi‐drug resistant (MDR) bacteria has prompted the substantial curiosity among the scientists to seek new tactics to combat the bacterial growth. Therefore, to eradicate the pathogenic bacteria with least cytotoxicity, we employed carbon dots as a broad spectrum of antibacterial weapons in the presence of visible light. Instead of using citric acid, we engaged the penicillin G as a carbon source for the synthesis of penicillin carbon dots (PCDs), which made the carbon dots more aggressive towards pathogenic microbes. Penicillin was also covalently attached to –NH2 containing citric acid based CDs (CDs‐Penicillin) via an amide bond to evaluate whether penicillin in the form of PCD has retained its activity or in its conjugated form (CDs‐Penicillin). Synthesized dots were assessed for their antibacterial activity against Staphylococcus aureus, Escherichia coli (DH5α), MDR Escherichia coli and Methicillin‐resistant Staphylococcus aureus in the presence as well as the absence of visible light. The mechanism of bacteria‐killing through cell wall rupturing was investigated using scanning electron microscopy. Antibacterial assay demonstrates that penicillin in the form of PCDs retained its activity and possess great prospects in the development of new bactericidal therapies to invade the MDR bacteria. Cytotoxicity of both PCDs and CDs‐Penicillin has been evaluated by measuring the viability of human HeLa cells. Fluorescence images of bacteria collected using different excitation wavelength.