Colorectal cancer (CRC) is the third most frequent cancer type in both males and females, with about 35% of patients being diagnosed in stage IV metastatic disease. Despite advancements in treatment, life expectancy in patients with metastatic disease is still not satisfying. Due to frequent drug resistance during conventional and targeted cancer treatments, the development and testing of multi-target therapies is an important research field. Medicinal mushrooms specific isolated compounds as well as complex extract mixtures have been studied in depth, and many mushroom species have been proven to be non-toxic multi-target inhibitors of specific oncogenic pathways, as well as potent immunomodulators. We established a significant antiproliferative and proapoptotic effect of medicinal mushroom preparations AGARIKON.1 and AGARIKON PLUS on non-colorectal (A549, MCF-7, MIA PaCa-2, HepG2) and colorectal (HCT-116, SW620) tumor cell lines, while the effect on human fibroblast cell line (WI-38) was proliferative. We further investigated the effect of medicinal mushroom preparations AGARIKON.1 and AGARIKON PLUS in various combinations with 5-fluorouracil in CT26.WT mouse model. Since the treatment started only after 14 days after tumor inoculation, this represents a very late tumor model of advanced metastatic cancer. The results show that AGARIKON.1 and AGARIKON PLUS exhibit immunostimulatory, antiangiogenic properties which affect survival differently: AGARIKON PLUS has the weakest effect on increasing overall survival (OS = 22.2%), in combination with 5- FU significantly prolongs it in relation to the negative control (OS = 87.5%), but not statistically significantly more in relation to 5-FU (OS = 77.7%) alone. The antitumor effects of AGARIKON.1 and AGARIKON PLUS, with or without 5-fluorouracil, are based on enhancing M1 macrophage polarization, inhibition of M2 and TAM polarization, effects on Th1/Th2/Th17 citokine profiles, direct inhibition of CT26.WT tumor growth, inhibition of vascular endothelial growth factors (VEGF) and metalloproteinases 2 and 9 (MMP-2 and MMP-9) modulation. Some of these effects can be attributed to the antioxidant ability of phenolic and other components present in medicinal mushrooms. The administration of AGARIKON.1 and AGARIKON PLUS did not show genotoxicity nor any adverse effect on clinical hematological or biochemical parameters. Furthermore, proteomic profiling of tumor tissues revealed that AGARIKON.1 and AGARIKON PLUS alone, or in combination with 5-fluorouracil, exert their antitumor effects by affecting fundamental processes in colorectal cancer (CRC) progression, such as translation, unfolded protein response (UPR) as well as lipid and tricarboxylic acid cycle (TCA). This data provides good basis for an expanded translational study.