Hydrogen sulfide (H2S) formed during sulfur metabolism in bacteria has been implicated in the development of intrinsic resistance to antibacterial agents. Despite the conversion of H2S to hydropersulfides greatly enhancing the biochemical properties of H2S such as antioxidant activity, the effects of hydropersulfides on antibiotic resistance have remained unknown. In this work, we investigated the effects of H2S alone or together with cystine to form cysteine hydropersulfide (CysSSH) on the activities of antibacterial agents. By using the disc diffusion test, we found that CysSSH treatment effectively inactivated β-lactams of the penicillin class (penicillin G and ampicillin) and the carbapenem class (meropenem). These β-lactams were resistant to treatment with H2S alone or cystine alone. In contrast, cephalosporin class β-lactams (cefaclor and cefoperazone) and non-β-lactam antibiotics (tetracycline, kanamycin, erythromycin, and ofloxacin) were stable after CysSSH treatment. Chromatographic and mass spectrometric analyses revealed that CysSSH directly reacted with β-lactams to form β-lactam ring-opened carbothioic S-acids (BL-COSH). Furthermore, we demonstrated that certain bacteria (e.g., Escherichia coli and Staphylococcus aureus) efficiently decomposed β-lactam antibiotics to form BL-COSH, which were transported to the extracellular space. These data suggest that CysSSH-mediated β-lactam decomposition may contribute to intrinsic bacterial resistance to β-lactams. BL-COSH may become useful biomarkers for CysSSH-mediated β-lactam resistance and for investigation of potential antibacterial adjuvants that can enhance the antibacterial activity of β-lactams by reducing the hydropersulfides in bacteria.