By means of carboxymethylation, a novel water-soluble carboxymethyl chitin-glucan (CM-CG) was prepared from the mycelium of Aspergillus niger, and its ability to stimulate macrophages was assessed and compared to that of the previously studied carboxymethylated glucan (CMG) from the yeast Saccharomyces cerevisiae. It was demonstrated that single intraperitoneal (i.p.) administration of CMG and CM-CG to the CBA mice led to a significant increase of leukocyte number. At the same time, the number of monocytes in the bone marrow was increased to more than two-fold. Application of both polysaccharides also resulted in the augmented number of liver macrophages and to the rise of their content of the secondary lysosomes. A markedly enhanced carbon clearance was observed as well as the increased release of tumor necrosis factor-α by the peritoneal macrophages indicating their amplified phagocytic activity. The effect of CM-CG in these experiments was ca. 1.7 times higher than that of CMG. Administration of both polysaccharides also led to the elevated level of free acid phosphatase in liver homogenate, implying labilization of the lysosomes. Increased serum chitotriosidase also indicated increased macrophage activity. The results obtained indicate similar in vivo macrophage stimulation activity of both applied fungal polysaccharides and suggest their potential clinical use as non-toxic natural compounds.