Bcl-xL is an apoptosis inhibitor that is upregulated in bladder cancer (BCa) and provides an attractive target for molecular therapies. Treatment with specific antisense oligodeoxynucleotides (AS-ODNs) or small interfering RNAs (siRNAs) were able to sensitize BCa cells to conventional chemotherapeutics. Ten new Bcl-xL-targeting AS-ODNs were systematically designed by using predicting software. AS-BX2034 and AS-BX2100 as well as the previously optimized siRNA construct si-BX713 were selected for further detailed in vitro analysis in the BCa cell lines UM-UC-3 and EJ28. Bcl-xL mRNA and protein expression levels, cell viability and apoptosis were examined 72 h after transfection. A single treatment with AS-BX2034 or AS-BX2100 caused only a low inhibition of the Bcl-xL mRNA expression with the highest reduction of ≤20% in UM-UC-3 cells. In contrast, a single treatment with si-BX713 strongly decreased Bcl-xL mRNA expression level by ≤69% in UM-UC-3 cells and by ≤86% in EJ28 cells. Both gene expression inhibitor types induced a low to moderate reduction of viability. Depending on the cell line, a combined treatment with AS-BX2100 or si-BX713 and cisplatin (CDDP) caused an additional inhibition of cell viability by ∼33 and 38%, respectively, compared to the respective control construct combined with CDDP. In comparison to the respective control treatment, combinations of AS-BX2100 and CDDP led to a stronger induction of apoptosis by 57% in UM-UC-3 cells and 44% in EJ28 cells, whereas the combination of si-BX713 and CDDP enhanced apoptosis by 38 and 118% in UM-UC-3 and EJ28 cells, respectively. Our comparative studies showed a stronger knockdown of Bcl-xL by the siRNA construct compared to AS-ODN treatment in both BCa cell lines. In combinatory treatments, the Bcl-xLdirected siRNA markedly enhanced the anti-proliferative and apoptotic effects of CDDP and therefore, may serve as suitable tool for chemosensitization of BCa cells.