Sugarcane has a high concentration of sucrose, which is metabolized by yeasts during ensiling producing high concentrations of ethanol. Calcium oxide (CaO) has been used as a silage additive resulting in a reduction of yeasts, less ethanol and improved DM recovery. The objective of this study was to evaluate the effect of CaO on the in vitro growth of lactic acid bacteria (LAB) and yeast in an aqueous-extract of sugarcane. The aqueous-extract of sugarcane was made by blending fresh sugarcane and distilled water in the proportion 5:1 for 5 min. The extract was filtered through layers of cheesecloth and sterilized at 121°C for 15 min. The microorganisms evaluated were one yeast (Saccharomyces cerevisiae), and 3 LAB (Lactobacillus brevis, L. plantarum, and Pediococcus pentosaceus, isolated from alfalfa silage). The LAB and the yeast (initial population of 6 log cfu/mL) were grown in the sugarcane extract with levels of CaO: 0 (control), 0.5, 1, 1.5, and 2% (fresh basis) and incubated at 37 and 30°C for 48 h, respectively. Two tests were conducted, one with no pH adjustment and another adjusting the initial pH to 6 ± 0.13. The pH was evaluated before and after the incubation. In the first trial, the final pH of the control was 5.62 whereas the pH of the extracts containing CaO were higher than 11.6 for all microorganisms. In addition, there was inhibition of growth of all microorganisms at the concentration of 0.5% CaO. In the second test, the numbers of L. brevis and P. pentosaceous decreased from 7.94 and 7.82 log cfu/mL to 4.97 and 5.98 log cfu/mL with adding 1% CaO and no growth was observed for all LAB at 1.5% CaO, except for L. brevis (3.75 log cfu/mL at 1.5% CaO). Numbers of S. cerevisiae remained constant in all concentrations of CaO (average 7.82 log cfu/mL). The results indicate that the initial rise in the pH caused by the addition of CaO delays the fermentation process and thus controls the growth of yeasts and ethanol production. In addition, other microorganisms other than those in the present study are responsible for lowering the pH in the early stages of fermentation.