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Lippia sidoides Chain. is a native shrub from the semi-arid region of Northeast Brazil. Its essential oil has high commercial value, due to the major compounds thymol and carvacrol, which have strong antimicrobial and antiseptic properties. The effect of concentrations and immersion time in sodium hypochlorite, culture media, the use of antibiotic and antioxidants on in vitro establishment of L. sidoides were evaluated. The assays were conducted in a completely randomized design. We evaluated the concentrations 0.2; 0.4; 0.6 and 0.8% of sodium hypochlorite and 8; 12; 16 and 20 minutes of immersion, in a 4 x 4 factorial scheme; the concentrations 0; 50; 100; 1.50 and 200 mg L-1 of cefotaxime sodium; the medium cultures MS, B5 and WPM; and the effect of antioxidants (PVP: 0.5 and 2 g L-1; and activated charcoal: 3 and 12 g L-1). The concentration of 0.8 % of sodium hypochlorite resulted in a significantly higher (p<0.01) number of leaves per shoot: 1.88. For the other characteristics we did not observe any significant effect of sodium hypochlorite concentrations: the contamination varied from 33.7 to 50.6%; the number of new shoots varied from 1. 17 to 1.65, and the number of leaves per explant varied from 1.77 to 3.07. Although we did not observe significant difference for immersion times, 12 and 16 minutes of immersion tend to result in minor contamination. Increasing the immersion time from 16 to 20 minutes tends to induce a reduction of new shoots (form 1.52 to 1.22), number of leaves per explant (from 2.62 to 1.81) and number of leaves per shoot (from 1.70 to 1.24). The use of cefotaxime sodium reduced significatively the bacterial contamination (55.23% at the control treatment; 9.99% at the 200 mg L-1 concentration), increasing the survival of explants from 0 (control) to 37.32% (200 mg L-1). The medium cultures offered statistically identical results. All the evaluated antioxidants, even at the lowest concentrations, reduced the oxidation from 50% (control) to as little as 10%. For in vitro establishment of L. sidoides nodal segments, the results indicate immersion of explants for 16 minutes in a 0.8% sodium hypochlorite solution, 200 mg L-1 of cefotaxime sodium, WPM, MS or B5 culture medium, and 3.0 g L-1 of activated charcoal or 0.5 g L-1 of PVP.