This study was carried out to investigate the prevalence of honeybee (Apis mellifera) diseases in Gwangju area. From November 2016 to August 2017, 89 samples were collected from 33 apiculture farms and reverse transcriptase-polymerase chain reaction (RT-PCR), polymerase chain reaction (PCR), and real time PCR were conducted. 14 infectious pathogens, including seven viruses, two bacteria, three fungi, and two parasites, were investigated from random apiculture farms in Gwangju. The percentage of infectious pathogens were as follows: Stonebrood (76.4%), Deformed wing virus (51.7%), Nosema (27.0%) in PCR and RT-PCR. This result indicated that Stonebrood was most prevalent disease in Gwangju area. And we could get similar results from real time PCR. 84.8% of farms have more than two of infectious pathogens. Stonebrood and Deformed wing virus were major diseases in almost all seasons and Black queen cell virus disease was especially prevalent in May.