Objective　To investigate the effect of andrographolide on enterovirus 71 (EV71) and coxsackievirus A16 (CA16) in vitro. Methods Cytopathic CPE assay and thiazolyl blue tetrazolium bromide (MTT) assay were used to determine the maximum non-toxic dose of andrographolide on RD cells and the inhibitory effect of andrographolide on EV71 and CA16 infection in vitro. The effects of andrographolide on the virus VP1 gene and cellular inflammatory factors (IL-1β, IL-6, and TNF-α) at gene levels were detected by quantitative fluorescence PCR. Results MTT results showed that the maximum non-toxic dose of andrographolide on RD cells was 78.00 μg/mL. Upon infection with hand-foot-mouth disease viruses, cells treated with andrographolide solutions at concentrations of 78.00, 39.00, 19.50, 9.75, 4.88, and 2.44 μg/mL showed increased survival rates to (82.41±1.76)%, (79.54±2.91)%, (81.02±1.99)%, (71.81±2.26)%, (52.87±1.51)%, and (50.41±0.93)% for EV71 and (81.00±0.64)%, (79.72±1.38)%, (61.59±3.47)%, (53.37±0.53)%, (52.41±1.37)%, and (43.69±0.40)% for CA16, respectively, indicating a significant reduction in the cytopathic effect caused by EV71 and CA16. When infected with hand-foot-mouth viruses and treated with andrographolide solutions at concentrations of 78.00, 39.00, 19.50, and 9.75 μg/mL, the expression levels of VP1 in EV71 and CA16, along with pro-inflammatory factors IL-1β, IL-6, and TNF-α in RD cells, were significantly lower compared to the virus control group. These results indicated that after infection with hand-foot-mouth viruses, treatment with andrographolide solution significantly inhibits the expression of the VP1 gene and reduces the mRNA levels of IL-1β, IL-6, and TNF-α. Conclusions Andrographolide exhibits obvious in vitro antiviral effects against EV71 and, potentially through the inhibition of the expression of pro-inflammatory factors IL-1β, TNF-α, and IL-6, thereby exerting its antiviral effects.