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Objective: We aimed to assess performance parameters in a Hawassa Tuberculosis Culture Laboratory, in the Sidama Regional Public Health Institute. Methods: A cross-sectional survey was conducted between 27 October 2020 and 31 May 2021, on 439 clinical specimens. The specimens were processed using standard procedures, and the final suspension was inoculated into a Microbial Growth Indicator Tube and Lowenstein–Jensen media slant. Ziehl–Neelsen staining and the Bioline test kit were used to identify and confirm Mycobacterium tuberculosis. The data were analyzed using the IBM Statistical Package for Social Sciences (SPSS, version 26). Results: Out of a total of 439 specimens that were processed, the recovery rates for smear-positive specimens were 61% (25 out of 41) and 58.5% (24 out of 41) for the Mycobacterial Growth Indicator Tube, and the Lowenstein–Jensen methods, respectively. For smear-negative samples, the recovery rates were 4.5% (18 out of 398) for both methods. Only 4 (0.9%) specimens were rejected. The mean turnaround times to detect mycobacteria from smear-positive samples were 14 and 32 days for the Mycobacterial Growth Indicator Tube and Lowenstein–Jensen methods, respectively. The standard deviations were ±6.3 days and ±9.7 days, respectively. For smear-negative samples, the mean turnaround times were 17.7 and 31 days for the Mycobacterial Growth Indicator Tube and Lowenstein–Jensen methods, respectively. The standard deviations were ±9.2 days and ±9.6 days, respectively. The contamination rates for the Mycobacterial Growth Indicator Tube and Lowenstein–Jensen methods were 9.8% (43 out of 439) and 9.6% (42 out of 439), respectively. The detection rate of nontuberculosis mycobacteria was 1.4% (6 out of 439). Conclusion: It demands attention to improve the low recovery rate among smear-negative cultures and culture contamination rates.