A delay in the diagnosis of tuberculosis (TB) is a public health problem in Japan. This delay may increase the chance of a TB patient visiting in a medical institution without a TB ward. We evaluated the practices of nurses for fit testing and seal checking their N95 respirator masks at hospitals without tuberculosis wards in Kanagawa, Japan. Of 36 nurses who participated in a medical course on infection control and medical safety in June 2010, 33 (91.7%) answered a questionnaire. Seven (22.6%) and 8 (26.7%) nurses had practical experience of fit testing and seal checking N95 masks, respectively. Nurses affiliated with hospitals having sanatorium wards were more likely to be acquainted with fit testing and seal checking than nurses from hospitals with only general wards [6 (39.9%) vs. 0 (0%) and 7 (46.7%) vs. 0 (0%), respectively]. Fewer than 30% of nurses exposed to TB patients had no experience with fit testing and scale checking N95 masks. Only one nurse had practical experience fitting testing at the hospital where she worked. Although the sample size in this study was small, these results showed inadequate experience in handling N95 respirator marks among Japanese nurses at hospitals without tuberculosis wards, which suggests the need to educate nurses practically in fit testing and seal checking N95 respirator masks.