The aim of this study was to determine the botanical origin, phenolics, flavonoids and fatty acids content, antioxidant and antimicrobial properties, of 18 bee collected pollen (BP) samples from Turkey and Romania. Five plant families with predominant pollens (>45%) were found in the Turkish samples, and only three in the Romanian samples. The phenolic content, varied between 16.40 and 41.17 mg GAE/g and the flavonoids content varied between 2.39 and 7.17 mg QE/g. The highest value of DPPH was 2.93 mmol Trolox/g and 9.64 mmol Trolox/g for the TEAC, whereas the IC50 value of alpha-Amylase inhibition was 8.10 mg/mL. We also verified that the presence of the methanolic extract of BP differentially affected the growth of Gram-positive and Gram-negative bacteria under study, strongly depending on the microorganism and the botanical origin of the BP samples used. The fatty acids contents were closely correlated with the above-mentioned parameters especially with the botanical origin and antibacterial activity. Our findings suggest that BP is a rich source of unsaturated fatty acids and bioactive compounds, which can be considered a value-added product. Furthermore, the differences in Turkish and Romanian BP chemical composition is also shown based on their antimicrobial and alpha-amylase inhibitory activities.