Protocatechuic aldehyde (PCA) is a natural phenolic acid compound with pharmacological effects such as anti-oxidative stress, antibacterial, anti-apoptotic, anti-inflammatory, anti-platelet aggregation, and anti-tumor. Despite the favorable therapeutic effects of PCA, it is imperative to recognize that adverse drug reactions can arise even with satisfactory quality assurance measures and during standard clinical application and dosing. Additionally, the acute toxicity and cardiotoxic sequelae of PCA are frequently under reported in the available documentation. To investigate the acute toxicity and cardiotoxic effects of PCA, the present study comprehensively assessed the acute toxicity and cardiotoxic effects of PCA by administering different concentrations of PCA and by monitoring the phenotypic changes in zebrafish, using AB wild-type Tg(cmlc2:EGFP) zebrafish as the experimental model organism. Meanwhile, the target genes of PCA that may cause cardiotoxicity were predicted and validated using a network pharmacology approach. Our findings indicated that PCA exhibited severe acute toxicity and cardiotoxic effects in zebrafish at 70 μg/mL and 80 μg/mL. Furthermore, PIK3CA, PARP1, and GSK3β may be involved in the mechanism of action of the cardiotoxicity-inducing effects of this compound. The present investigation has afforded a deeper insight into the acute toxicity and cardiotoxic impacts of PCA on zebrafish and has established a significant theoretical foundation for the evaluation of toxicity in pharmaceuticals incorporating PCA.