Lentinus edodes (shiitake) ranks among the most well-known medicinal mushrooms worldwide. Immune-modulating effects of shiitake extracts have been widely demonstrated in animals and humans. Apart from providing highly purified compounds such as lentinan, crude mushroom extracts also show antitumor activities. The direct cytotoxicity of crude preparations still requires investigation. The focus of our study lies in the molecular weight cutoff distribution of β-glucans and proteins in a cold aqueous extract from L. edodes. We applied cross-flow ultrafiltration to obtain 6 fractions with different molecular sizes. β-glucan and protein contents were quantified. We were able to show that only small amounts of β-glucans were extracted and that protein content decreases with fraction size. The cytotoxic potential of the cold aqueous preparation was demonstrated by analyzing inhibition of the growth of non-small cell lung cancer and triple-negative breast cancer cells.