<p>Evaluation of measurement uncertainty is a critical requirement for laboratories accredited to ISO/IEC 17025, particularly in the specialized nutrition industry where rigorous microbiological safety criteria protect vulnerable populations. However, quantifying uncertainty in quantitative microbiological testing faces unique challenges due to biological variability and matrix heterogeneity, rendering the classical bottom-up approach impractical. This Practitioner’s Report presents the development and application of a practical Reference Guide based on the holistic top-down strategy recommended by ISO 19036. While the standard normatively permits the use of either interlaboratory study data or internal replication matrixes, this guide focuses strictly on the intralaboratory pathway. The guide uses the pooled intra-laboratory reproducibility standard deviation to evaluate combined standard uncertainty. The operational framework was verified through a case study for the enumeration of molds (<i>Aspergillus brasiliensis</i>) in skimmed milk powder. Results demonstrated an expanded uncertainty of 0.2 log₁₀ CFU/g. To facilitate practical compliance interpretation at borderline zones, this log-uncertainty is mathematically reconverted at the critical control threshold (&lt; 10&#xa0;CFU/g), yielding an asymmetrical linear operational interval of 6.3 to 15.8&#xa0;CFU/g. This approach successfully aligns the measurement uncertainty profile with routine industrial manufacturing specifications. This work provides a traceable operational roadmap for industrial testing facilities, ensuring regulatory compliance and measurement reliability in commercial release decisions for sensitive product batches.</p>