Like many traditional Chinese herbal medicines, preparations from
Radix Dipsaci
are at risk of contamination by harmful mycotoxins; however, there have been no reports of actual contamination. In this study, we developed an analytical method to simultaneously detect eight mycotoxins in
Radix Dipsaci
and estimate the exposure risk for consumers. We have developed an analytical method utilizing ultra-high performance liquid chromatography and tandem mass spectrometry to accurately determine the levels of AFB1, AFB2, AFG1, AFG2, OTA, ZEN, T-2 and ST mycotoxins in 45 batches of
Radix Dipsaci
sourced from major medicinal herb markets across five regions in China. We also analyzed migration of mycotoxins from the raw herbs into water decoction. Based on these results and data on human consumption of the herbal medicine, we estimated risk of exposure and acceptable exposure limits to mycotoxins in the
Radix Dipsaci
using the “margin of exposure (MOE)” method. Of the 45 batches of
Radix Dipsaci,
48.89% contained at least one of the eight mycotoxins, 24.44% contained one, 17.78% contained two and 6.67% contained three. The most frequent mycotoxins were aflatoxin B1, present in 35.56% of batches (at 0.25–34.84 μg/kg); aflatoxin G1, 15.56% (1.99–44.05 μg/kg); and ochratoxin A, 22.22% (16.11–143.38 μg/kg). These three mycotoxins transferred from the raw herb into water decoction at respective rates of 20.20%, 29.14%, and 24.80%. The 95th percentile values of the MOE risk factors for health effects of AFB1 were below 10,000 at high doses but above 10,000 at low doses of
Radix Dipsaci
long-term treatment. With the reduction in duration of exposure years, the MOE values of AFB1 and AFG1 gradually reverted to within the acceptable range. The mean, 50th, and 95th percentile values of the MOE risk factors for health effects of OTA exceeded 10,000 regardless of whether consumers received a low or high dose of
Radix Dipsaci
treatment for durations ranging from 1 to lifetime. Based on this exposure and a typical human diet, we have estimated the respective 20-year exposure limits for
Radix Dipsaci
to be 5.821 μg/kg, 4.035 μg/kg, and 56.073 μg/kg for the three mycotoxins under consideration. Contamination with multiple mycotoxins is frequently observed in
Radix Dipsaci
, and the three most prevalent contaminants have been found to leach into water decoctions, thereby posing a potential health hazard for individuals consuming this herbal preparation. This work highlights the need to monitor herbal medicines for mycotoxin contamination in order to protect consumers.