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Liver fibrosis is a wound-healing response to chronic liver injury that could lead to liver failure, but treatment remains ineffective. In this study, we investigated anti-hepatic fibrosis activity of n-hexane, chloroform, ethyl acetate, and methanol extracts of mycelia from six commercially available medicinal mushrooms in submerged culture, namely Antrodia camphorata, Cephalosporium sinensis, Cordyceps mortierella, Hericium erinaceus, Ganoderma lucidum, and Armillaria mellea. Their anti-fibrotic activities were evaluated via inhibition against accumulation of TGF-β1-induced collagen deposition in CFSC-8B cells. Hex, Chl, and MeOH extracts of A. camphorata and Hex extract of A. mellea significantly decreased collagen production. Bioactivity-guided fractionation led to the identification of seven compounds using UPLC-Q-TOF-MS from the Hex Fr.2 of A. camphorata. At the molecular level, Hex Fr.2 of A. camphorata suppressed α-SMA, Collagen I, Collagen III, and Fibronectin expression induced by TGF-β1 in CFSC-8B cells as indicated by qRT-PCR analysis. They also inhibited α-SMA and Collagen I protein expression according to western blot analyses. Mechanistically, Hex Fr.2 of A. camphorata negatively regulates TGF-β1/Smad2/3 signaling. Our studies demonstrate that A. camphorata has in vitro anti-hepatofibrotic activity and that there is great potential for the discovery of new drugs for the treatment of liver fibrosis by screening more medicinal mushrooms.