Bee pollen is a product from beehives that is contained in corbiculae, generally monospecific and is currently considered as a functional food due to its nutritional and medicinal properties. This study investigates the antimicrobial activity of a Chilean bee pollen extract of known botanical origin. The botanical origin of the pollen was determined by palynological analysis, and then an aqueous extract was prepared. The antibacterial properties of the extract were evaluated on human infectious agents (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pyogenes) using a qualitative method (agar diffusion) and a quantitative method (minimum inhibitory and bactericide concentration) and on agricultural pathogens (Alternaria alternata, Botrytis cinerea and Fusarium oxysporum) using the poisoned food technique. The bee pollen (60% of Azara petiolaris) is considered native, endemic and monofloral. The E. coli and P. aeruginosa became resistant to the extract, while S. aureus and S. pyogenes were sensitive. Additionally, the minimum inhibitory concentrations (MICs) of these bacteria were 82.4 mg mL-1 for E. coli, 41.2 mg mL-1 for P. aeruginosa, and 20.6 mg mL-1 for S. aureus and S. pyogenes. Additionally, the extract did not have a complete inhibitory effect on the fungi, but it caused delayed growth in comparison to the control. The development of an aqueous extract from native and endemic bee pollen with antimicrobial properties creates the potential for future research and development of new Chilean natural products, favoring the development of national apiculture.