Thirty-five different lactic acid bacteria (LAB) strains were screened for their conjugated linoleic acid (CLA) isomers (C18:2 cis-9, trans-11 and C18:2 trans-10, cis-12) producing ability from linoleic acid (LA) in sheep’s milk. Preliminary experiments revealed that Lactobacillus delbrüeckii ssp. bulgaricus 2230 and Streptococcus thermophilus St 360 among the screened strains had the highest CLA-producing ability. This two strains were assayed in an 11-run fractional factorial design to investigate the effect of four variables included glucose, powdered sheep milk, LA and inoculum ratio on CLA production in a sheep’s milk yogurt. The optimum conditions for producing the highest levels of CLA (42.86%) were obtained by adding 10.00 mg/mL of glucose, 30.00 mg/mL of powdered sheep milk, 0.90 mg/mL of LA and a 1:2 (St:Lb) ratio of bacterial strains in the inoculum. This CLA-rich sheep’s milk yogurt could be an important supplementary food source for increasing the CLA in the human diet.