Cephalosporins, derivatives of 7-aminocephalosporanic acid (7-ACA), are potent antibacterial agents. The toxicity prediction of these compounds is of considerable importance in new drug development. Zebrafish embryo toxicity testing was thought to be suitable for evaluation of the toxic properties of cephalosporins. Here, five kinds of cephalosporins and their isomers were used for investigation of the toxic functional groups of cephalosporins and for further evaluation of the efficacy of zebrafish embryo toxicity testing. Computational chemistry methods were also used to study the conformations of the stereoisomers of cephalosporins in aqueous solution to explore the relationship between the stereoisomers and the experimental results of toxicity tests on zebrafish embryos. Our results suggest that both the C-7 and C-3 substituents of cephalosporins are toxic functional groups. The toxic functional groups increase the toxic reaction of 7-ACA and can induce variable abnormal phenotypes in zebrafish embryo toxicity testing. The embryonic toxicities of cephalosporins were involved in organogenesis, mainly in the development of the cranial nerve, cardiovascular system, notochord and abdomen, and pigment formation; those tissues and organs are derived from ectoderm, mesoderm, and endoderm. The theoretical calculations showed a strong negative correlation between topological polar surface area (TPSA) values and the toxic effect, which indicated that molecular polarity may be crucial to the toxic effects of the isomers of cephalosporins. The concept of toxic functional groups may help us understand the safety differences of cephalosporins.