Ganoderma lucidum is a Chinese traditional medicine with various bioactivities. However, the impacts of a Ganoderma lucidum mycelium-fermented liquid (GLFL), as a promising alternative product, on the gut microbes and cardiovascular risk factors have not been explored in humans to date. In this study, the composition of polysaccharides in GLFL was analyzed, and eight volunteers were fed with GLFL to investigate its influence on gut microbes and cardiovascular risk factors. High-throughput 16S rRNA gene sequencing technique was utilized to investigate the effects of GLFL on the diversity of the bacterial communities and the composition of gut microbiota. The results demonstrated that GLFL significantly altered β diversity and several phyla of gut microbiota in volunteers. Moreover, GLFL provided protection to humans by promoting the growth of probiotics, incorporating genus Lactobacillus (p < 0.05), and reducing pathogens containing genus Campylobacter and Aggregatibacter (p < 0.05); however, GLFL also had detrimental effects as it increased the population of Firmicutes/Bacteroidetes and opportunistic pathogens including genus Acinetobacter, Pseudomonas, Serratia, Stenotrophomonas, Peptococcus and S24-7 (p < 0.05) and reduced that of probiotics genus Lactococcus. Finally, we found that GLFL reduced plasma low-density lipoprotein cholesterol (LDL-c) in volunteers. This is helpful for further understanding the effect of Ganoderma lucidum or its related products on human body.