Honeybee pollen loads result from the agglutination of pollen grains and salivary secretions of bees. The potential use of honeybee pollen as a food supplement greatly depends on its chemical composition, which varies depending on the botanical and geographical origin of the pollen grains. This study aimed to characterize the botanical origin, chemical composition, and antioxidant and antibacterial activities of honeybee pollen from the V Region of Chile. The introduced species Brassica rapa and Eschscholzia californica predominated in the bee pollen analyzed. The honeybee pollen extracts showed antioxidant and antibacterial properties, specifically against the pathogenic microorganism Streptococcus pyogenes. Quercetin and myricetin were found in all samples in large concentrations. The separation of pollen loads from a multifloral sample demonstrated that E. californica pollen loads are responsible for antibacterial activity. This sample also showed a high concentration of quercetin (304.8 mg/100 g of bee pollen). Based on the present results, honeybee pollen from the V Region of Chile has been found to exhibit antioxidant and antimicrobial activities. Furthermore, it is proposed to use quercetin as a quality indicator for honeybee pollen from this region of Chile. These results should help establish better quality control criteria for Chilean honeybee pollen and its potential use as a functional ingredient.