A comparative study is performed on Hypericum perforatum (H. perforatum) and doxorubicin (Dox) anticancer agents. Double emulsion and sonication techniques were used to enhance drug loading efficiency in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). To distinguish the efficiency of the double emulsion method, drug entrapment efficiency was measured. In vitro release studies were carried out to evaluate drug release profiles. Entrapment efficiency was estimated to be 48 and 21% for Dox-loaded and H. perforatum-loaded NPs, respectively. Surprisingly, the encapsulation process disrupted the formation of Dox crystals (Dox in NPs converted from the crystalline to the amorphous phase), whereas disordered crystalline was observed for H. perforatum. In vitro release studies suggested that the total released drug was about 71% of the whole entrapped drug after 20 days. MTT assay confirmed that Dox was more toxic than H. perforatum. This study revealed that H. perforatum is a good choice for further experiments in drug delivery systems because of low cardiotoxicity according to the herbaceous nature of this anticancer agent.