Sinop is one of the chestnut growing regions of Turkey. The pollen of Castanea sativa is consisting of the main source of honeybee. In the present study were investigated to phenolic composition, antioxidant and antimicrobial efficiency of methanolic extracts of C. sativa pollen grains collected from 9 different populations during inflorescence period (July, 2013). The extracts were screened for their antimicrobial activities against to the 8 bacteria and 3 yeast species by using disc-diffusion procedure. The pollen extracts displayed more effective against to Gram-positive bacteria rather than Gram-negative, the extracts had also moderate anti-yeast activity. In vitro the antioxidant activity based on the 1,1-diphenly-2-picryl hydrazyl (DPPH) free radical was evaluated for all the extracts, and it was found that the extracts had good antioxidant activity in the range of the IC50= 0.093-19.5 mg mL-1. The total phenolic contents of the extracts were determined between 64.02 ± 0.26 mg GAE g-1 and 103.8 ± 6.72 mg GAE g-1. In addition, we determined a negative linear correlation (r= - 0.59) between the total phenolic content and IC50 values. In this sense, our findings confirmed that the methanolic pollen extracts had good antioxidant activity. In conclusion, the result of the study suggested that Castanea sativa Mill. pollen grains can be a good natural product source of antioxidant and antimicrobial agents for potential health benefits.