Bee pollen is produced by honeybees from the agglutination of pollen grains belonging to one or more plant species. Although it is intended to be a nutritional source for the hive, its remarkable concentration of nutrients and phytochemicals, combined with its pleasant organoleptic qualities, makes it appealing for human consumption. This study examined the phytochemical content and the antioxidant and antimicrobial activities of a polyfloral bee pollen collected in Tuscany (Italy). Additionally, its less studied anti-inflammatory potential towards tumor necrosis factor-alpha (TNF-α)-inflamed A549 cells was evaluated to assess its possible use in inflammatory respiratory diseases. Bee pollen extract (BPE) was chemically characterized in terms of total polyphenol (20.2 ± 1.5 mg gallic acid equivalents/g fw), flavonoid (9.22 ± 0.64 mg quercetin equivalents/g fw), and carotenoid (10.4 ± 1.4 µg carotenoids/g fw) contents. BPE exerted good antioxidant and antiradical activities in ferric reducing antioxidant power (38.6 ± 4.5 mg Fe