In this report, we describe the first three cases of Tsukamurella conjunctivitis in the literature. All three patients presented with congestion of one eye with small amounts of serous discharge for 1 to 2 days. All three recovered after 10 days of treatment with polymyxin B-neomycin or chloramphenicol eyedrops. Sequencing of the 16S rRNA genes of the three isolates recovered from the serous discharge of the three patients showed that they were all Tsukamurella species. The phenotypic characteristics of the isolate obtained from one patient best fit the phenotypic profile of Tsukamurella pulmonis, whereas those of the other two best fit that of Tsukamurella tyrosinosolvens.