Paenibacillus larvae is one of the major bacterial pathogens of honey bee broods and the causative agent of American foulbrood disease. The factors responsible for the pathogenesis of American foulbrood disease are still not fully understood, and the increasing resistance of P. larvae to commonly used antibiotics necessitates a search for new agents to control this disease. The in vitro antibacterial activities of 28 plant essential oils against P. larvae ATCC 9545 were evaluated. Out of the 28 plant essential oils tested, 20 were found to be effective in killing P. larvae. Based on their minimum bactericidal concentration (MBC) values, the effective oils were grouped into three categories: highly effective, moderately effective and minimally effective. Jamaica pepper oil, mountain pepper oil, ajwain oil, corn mint oil, spearmint oil, star anise oil, nutmeg oil and camphor oil were highly effective, with MBC values between 162.0 and 375.0 mg/mL. Jamaica pepper oil was the most effective essential oil, with an MBC value of 162.0 mg/mL. The results of the time-response effect assays showed that no viable P. larvae cells were observed after 24 h of treatment with Jamaica pepper oil (162.0 mg/mL), 36 h of treatment with mountain pepper oil (186.0 mg/mL), 48 h of treatment with ajwain oil (224.8 mg/mL) or 48 h of treatment with oxytetracycline (5.89 mg/mL). The tested essential oils exhibited significant antimicrobial activities against P. larvae, and they may contain compounds that could play an important role in the treatment or prevention of American foulbrood disease.