Heat-clearing herbs (HCHs) have been widely used as single drug or adjuvant therapy in cancer treatment. However, it is unclear whether HCHs induce hormetic effect in cancer cells or not. Thus, the aim of this study is to examine hormetic effect of aqueous extracts of HCH and their antagonistic effect on anticancer activity of chemotherapeutic agents. In the first part, we found that the aqueous extracts of HCH (Cortex phellodendri, Hedyotis diffusa, Houttuyniae herba, Polygoni cuspidati, Paeoniae radix, Prunella vulgaris, Scutellaria baicalensis, Sophorae flavescentis and Taraxacum mongolicum) remarkably induced hormesis in B16-F10 murine melanoma cells. Low concentrations of aqueous extracts of P. vulgaris (APV) (0.32 and 0.63 mg/mL) and S. baicalensis (ASB) (0.32 and 0.63 mg/mL) increased cell viability and attenuated apoptosis induced by camptothecin (CPT) or cis-platinum (DDP), while high concentrations of APV (5 mg/mL) and ASB (2.5 mg/mL) shown significant anticancer effect alone or in combination with chemotherapeutic agents. Treatment with APV and ASB could promote the production of reactive oxygen species (ROS) in B16-F10 cells. In addition, low concentrations of APV and ASB upregulated the PI3K/Akt/FOXO3a pathway and increased the expression of antioxidase (CAT, GSH-Px and SOD2) as well as the activities of antioxidase (CAT, GPX1 and T-SOD) in B16-F10 cells. However, high concentrations of APV and ASB showed adverse effects. The PI3K inhibitor, LYP294002, attenuated the increasing effects of APV and ASB. In the second part, we found baicalein, an active component of S. baicalensis, induced hormetic effect in B16-F10 cells, MDA-MB-231 human breast cancer cells and A549 non-small cell lung cancer cells. Low concentrations of baicalein (0.4, 0.8 and 1.6 μM) antagonized anticancer effect of CPT and DDP in B16-F10 cells and activated the PI3K/Akt and MAPK pathways, while 25 μM baicalein shown anticancer effect alone or in combination with chemotherapeutic agents. Treatment with inhibitors of ERK1/2 and PI3K pathways attenuated the hormetic effect induced by baicalin and restored the anticancer activities of CPT and DDP, suggesting that ERK1/2 and PI3K/Akt pathways played important roles in the hormetic effect of baicalein. In summary, our results suggested that HCH extracts could induce hormetic effect and antagonize the antitumor activity of chemotherapeutic agents, CPT and DDP, in B16-F10 cells. Therefore, we need to assess the risk of HCHs before practicing in clinical trials.