Drugs against bacteria and abnormal cells, such as antibiotics and anticancer drugs, may save human lives. However, drug resistance is becoming more common in the clinical world. Nowadays, a synergistic action of multiple bioactive compounds and their combination with smart nanoplatforms has been considered an alternative therapeutic strategy to fight drug resistance in multidrugresistant cancers and microorganisms. The present study reports a onestep fabrication of innovative pH-responsive Janus nanofibers loaded with two active compounds, each in separate polymer compartments for synergistic combination therapy. By dissolving one of the compartments from the nanofibers, we could clearly demonstrate a highly yielded anisotropic Janus structure with two faces by scanning electron microscopy (SEM) analysis. To better understand the distinctive attributes of Janus nanofibers, several analytical methods, such as X-ray diffraction (XRD), FTIR spectroscopy, and contact angle goniometry, were utilized to examine and compare them to those of monolithic nanofibers. Furthermore, a drug release test was conducted in pH 7.4 and 6.0 media since the properties of Janus nanofibers correlate significantly with different environmental pH levels. This resulted in the on-demand sequential codelivery of octenidine (OCT) and curcumin (CUR) to the corresponding pH stimulus. Accordingly, the antibacterial properties of Janus fibers against Escherichia coli and Staphylococcus aureus, tested in a suspension test, were pH-dependent, i.e., greater in pH 6 due to the synergistic action of two active compounds, and Eudragit E100 (EE), and highly satisfactory. The biocompatibility of the Janus fibers was confirmed in selected tests.