Information regarding to microbiological and chemical characteristics, and incubation time is crucial in developing kefir prepared using Bali cattle milk. This study was intended to investigate microbiological and chemical properties of the kefir prepared of Bali Cattle milk and Indonesian kefir grains after 24, 48 and 72 hours incubation periods. A completely randomized design, with 3 treatments, and 9 replicates were undertaken. Kefir samples were taken at the end of incubation period for determination of total lactic acid bacterial and yeast counts, pH, titratable acidity, lactose percentage and protein content. The result of this research showed that the total lactic acid bacterial counts were 108 – 109 cfu/ml, while yeast counts were ranging from 105 – 106 cfu/ml, no coliform and Escherichia coli were detected in any kefir samples in this research. Identification of the lactic acid bacteria and yeast revealed that the Lactobacillus paracasei ssp. paracasei 1 was the predominant species found in the kefir samples, followed by Lactobacillus brevis and the yeast Candida famata. Chemical analysis of the kefir samples showed that, protein, lactose, titratable acidity and pH of the kefir samples were in the range of 5.68 - 6.26%, 3.98 - 4.67%, 0.89 - 1.73% and 3.38 - 4.35, respectively. The result also indicated that the incubation period significantly affected the microbial counts and chemical composition of Bali Cattle milk kefir. Keywords: Kefir, Bali cattle milk, Lactic acid bacteria, Yeast, Protein, Lactos