Salmonella species is one of the major bacterial agents that causes gastrointestinal disease in pigs. Natural antimicrobials derived from plants may be alternative therapeutics that could replace currently used antibiotics in the control of infectious disease. In this study, we assessed the antibacterial activity of Oenothera biennis L. extract against Salmonella Typhimurium both in vitro and in vivo. O. biennis L. extract had a strong inhibitory effect on S. Typhimurium in vitro, reducing bacterial growth by 87%. For the in vivo experiment, 16 post-weaned pigs were divided into 4 groups consisting of 4 pigs each: an uninfected, untreated negative control group; an untreated positive control group that was infected with S. Typhimurium; and two groups that were infected with S. Typhimurium and treated with either 0.1% or 0.5% O. biennis L. extract. Pigs were followed for 21 days after infection, and their body weight, daily gains, feed conversion ratio (FCR), and feed efficiency (FE) were monitored. The pigs treated with O. biennis L. extract had significantly higher daily gains than the positive control group (p<0.05). The O. biennis L. extract-treated pigs also exhibited better weight gain and FE, as well as a lower FCR and less severe diarrhea, than the positive controls but these results were not statistically significant. Our study demonstrates that O. biennis L. extract has antibacterial effects against S. Typhimurium, both in vitro and in vivo, and that these antibacterial effects may produce better growth performance in pigs infected with S. Typhimurium.