The EWS-Fli1 fusion gene, resulting from a t(11;22) translocation, plays a key role in the Ewing’s sarcoma pathogenesis. In the past, a 25mer phosphorothioate antisense oligodeoxynucleotide, a structured 30mer phosphorothioate/ phosphodiester antisense oligodeoxynucleotide, and an antisense siRNA, delivered either free, by vectors or intracellularly expressed, were found potent in various in vitro and in vivo Ewing sarcoma models. Because of differences among the models used in the literature, the comparison of various antisense agents with each other is difficult. Therefore, we aimed to evaluate these three antisense agents in NIH/3T3 fibroblasts which stably express the human EWS-Fli1 oncogene as an in vitro model of Ewing sarcoma. Fours parameters were considered including oncogene EWS-Fli1 and EWS mRNA expression, cellular proliferation, and actin cytoskeleton organization. They illustrate the antisense efficacy, the specificity and the phenotypic reversion for the last two ones, respectively. We showed that the structured 30mer phosphorothioate/phosphodiester antisense oligodeoxynucleotide and antisense siRNA represent the best choice for clinical trials. Nevertheless, the antisense ODN is more specific than the siRNA and represents the most efficient antisense agent. Its activity may be improved after the selection of an appropriate delivery vector which is able to increase cell penetration and to protect it from nucleases degradation.