Wild-grown fruiting bodies of the basidiomycete Fomitopsis betulina (Agaricomycetes, birch bracket mushroom, = Piptoporus betulinus) in different growing stages were collected and analyzed for their beta-glucan content. It could be shown that no significant difference in beta-glucan content regarding size or location of the collected fruiting bodies could be determined, but all samples displayed high values of beta-glucan in comparison to other well-known culinary or medicinal mushroom species. Furthermore, F. betulina fruiting bodies extracted with cold sodium chloride were separated into several fractions by cross flow ultrafiltration, and glucan and protein content were analyzed. The fractions showed varying amounts of beta-glucan and very low protein contents were detected. Also, bioactivity of the fractionated extract was analyzed. None of the mushroom extract fractions induced significant cytotoxicity after 48 h of incubation at a concentration up to 1 mg/mL. Interestingly, in a scratch wound assay, the extract FbS 1, an ultrafiltrated fraction > 300 kDa, was able to block tumor cell migration by 38% compared to solvent control after 48 h of incubation at a concentration of 0.33 mg/mL. In conclusion, our results have high potential for identifying novel antitumor activities based on F. betulina.