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National Tuberculosis Reference Laboratories (NTRLs) are central to tuberculosis (TB) control programs. Between 2018 and 2025, the Republic of Congo, a country of 6 million inhabitants, achieved a transformative strengthening of its TB diagnostic system, coordinated by the NTRL. Strategic investments, supported mainly by international partners, enabled a substantial decentralization of services, expanding the diagnostic network from 40 to 113 facilities and increasing GeneXpert sites from 3 to 31. This was bolstered by a national sample transport system that slashed diagnostic inequities and a robust External Quality Assessment framework to ensure reliability. Critically, the establishment of a BSL-3 facility and the deployment of advanced assays like Xpert MTB/XDR ended the reliance on overseas testing by introducing in-country capacity for multidrug-resistant and extensively drug-resistant TB detection. These systemic improvements directly translated into significant outcomes including an annual molecular testing surging from 11,609 to over 27,000, bacteriological confirmation rates rose from 34 to 73%. This comprehensive laboratory systems strengthening, which also facilitated cross-programmatic initiatives like HIV, Mpox testing integration, underscores how sustained investment in infrastructure, logistics, and quality management is fundamental to improving case detection, surveillance, and progress toward the WHO End TB Strategy milestones.