An adequate concentration of growth regulators as well as the replacement of agar by an alternative medium may be promising from practical and financial points of view to produce orchid plants by micropropagation. The objective of this work was to evaluate different concentrations of growth regulator and alternative substrates for agar replacement in culture medium for in vitro multiplication and rooting of Oncidium baueri. In the explant multiplication phase, two experimental factors were evaluated- various concentrations of 6-benzylaminopurine (BAP) (0.0, 1.0, 2.0, and 3.0 mg L-1) and substrates (agar, vermiculite, and coconut fiber) added to MS medium. In the rooting phase, different concentrations of indole butyric acid (IBA) (0.0, 0.5, 1.0, and 1.5 mg L-1) were added to culture medium containing the same substrate. Six months after the experiments were initiated, the survival percentage, number of leaves, shoots, and roots and length of the aerial part and the major root were evaluated. The results suggested that addition of 1.0 mg L-1 BAP is necessary for the O. baueri in vitro multiplication phase, but IBA is not necessary in the rooting phase. For the substrate, vermiculite is not indicated as an agar replacement. In contrast, coconut fiber can be used in both multiplication and rooting phases of Oncidium baueri in vitro culture.