ABSTRACT The honeybee (Apis mellifera L.) is an important pollinator worldwide. Beekeepers have experienced an increasing loss of colonies in recent years and pathologies including viruses and intestinal parasites such as Nosema ceranae are among those held responsible for this decline. The aim of this study was to determine the prevalence and distribution, using the real time PCR method, of six honeybee viruses including Deformed Wing Virus (DWV), Black Queen Cell Virus (BQCV), Sacbrood Virus (SBV), Acute Bee Paralysis Virus (ABPV), Chronic Bee Paralysis Virus (CBPV) and Israeli Acute Paralysis Virus (IAPV) in addition to three intestinal parasites, N. ceranae, N. apis and Lotmaria passim in Chilean beehives distributed between the Metropolitan Region and La Araucanía Region. Among the viruses, BQCV was the most prevalent in the analyzed hives, ranging from 72% to 100%, N. ceranae was detected between 19% to 53% and L. passim showed infections ranging from 45% to 73%. Similarly, BQCV (96%) and N. ceranae (42%) were more prevalent in spring-summer whilst DWV (37%) and L. passim (68%) were more frequents in autumn-winter seasons. Furthermore, mixed infections were also detected, wherein viruses were found along with L. passim or viruses with N. ceranae and L. passim, which proved to be the most prevalent. Of the 467 hives analyzed, 97.2% were infected with one or more of the studied pathogens, indicating that they are widely spread in Chile.