Human embryonic stem cells (hESCs) are an attractive cell source for cell-based therapy considering their unique characteristics of self-renewal and pluripotency. Optimal culture condition of hESCs is important for the continuous maintenance and differentiation of hESCs. For the elimination or prevention of mycoplasma contamination, antibiotics are used for routine culture of various cell lines, however, their effects on the differentiation of hESCs were not clarified. In this study, we tried to demonstrate the effects of mycoplasma removal agent, plasmocin, on undifferentiated hESCs and derived differentiating cells. There were no significant effects on the morphology and the expression of Oct4, Sox2 and Nanog in undifferentiated hESCs. However, cardiogenic potential of hESCs were significantly affected. The expression of cardiac lineage markers such as Nkx2.5, cTn I and Tbx5 was downregulated in plasmocin group. No dose-response relationship was observed. These results imply that differentiation of hESCs into cardiac lineage can be affected by antibiotics. Therefore, careful selection of infection prevention agent is necessary.