Information, Communication Technology (ICT) has become the order of the day. Globally, there is increasing quest for use of ICT in various spheres of life. The Health care sector is not left out: Computer based diagnosis is the hope of fast and accurate diagnostic process. GeneXpert machines for rapid diagnosis of Tuberculosis (TB) and drug resistant tuberculosis (DR-TB), work with GeneXpert (GX) software and computer programs. This study was carried out to assess Knowledge, Attitude and Practice of Laboratory staff on computer with the view to unraveling its role in scaling up Xpert MTB/Rif in Nigeria. The survey was done using a structured, closed-ended questionnaire administered to laboratory staff operating GeneXpert machine, who participated in the study. A total of 76 GeneXpert machine operators (56.7%) out of 134 laboratory staff trained from 31 Xpert sites in Nigeria were interviewed. These included 49 Laboratory Scientists, 15 laboratory technicians and 12 other laboratory staff that operate the machine. Majority, 55 (72.4%) of the respondents had good knowledge of computer; 43 (78.2%), 4 (7.3%) and 8 (14.5%) of these were laboratory scientists, technicians and other laboratory staff respectively. Good computer knowledge was highest among scientists and lowest among technicians. These differences were statistically significant (df = 1 P < 0.01). Age, gender, owning a personal computer and formal computer training significantly influenced computing knowledge. Most Xpert MTB/RIF users 45 (64.5%) had positive attitude towards computing and this was significantly influenced by respondent's age and formal computer training. Only 38 (50%) had good computing practice; this was significantly associated with owning a personal computer (P < 0.01) and formal computer training. The major computer operation challenges observed among the laboratory staff included; Xpert calibration; completion of electronic recording tool and software operations like importing of assay definition file; plunger maintenance; generating system and error log reports as well as archiving/retrieving of tests. Introduction of basic computer training module into the Xpert training curriculum, strict adherence to SOP, continuous supportive supervision and mentorship training are recommended in Nigeria to boost efficiency of laboratory staff.