The main active ingredient of Allium ascalonicum volatile oil was the onion capsaicin, the objective of this study was to investigate the antibacterial and antiviral effect of onion capsaicin extracted from Allium ascalonicum. The minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of onion capsaicin on Staphylococcus aureus and Escherichia coli were determined by the tube dilution method and antibacterial ring test. According to the cytopathic effect inhibition assay and neutral red staining method, the median toxicity concentration (TC50), maximum non-toxic concentration (TC0), median inhibitory concentration (IC50) and therapeutic index (TI) were calculated by Reed-muench analysis to determine its role in anti-respiratory syncytial virus (RSV). The results showed that onion capsaicin had some anti-gram positive bacteria and anti-gram negative bacteria effect in vitro. The TC50 and TC0 of onion capsaicin for HEp-2 cells were 6.3 μg/mL and 3.6 μg/mL, respectively. The IC50 and TI of onion capsaicin for RSV virus were 1.7 μg/mL and 3.71, respectively. Onion capsaicin could dose-dependently improve cytopathic effects caused by RSV infection and inhibit the proliferation of the virus. These results will provide a solid scientific basis for the further development and clinical application of onion capsaicin.