Microbial contamination may compromise the efficacy and safety of pharmaceutical products. Microbial enumeration tests are required by most of the pharmacopeial compendia and consist in conventional pour-plate inoculation of a sample aliquot followed by incubation under appropriate conditions. Despite of this, the measurement uncertainty evaluation for microbial enumeration tests is rarely considered. Thus, the aim of this paper was to assess the matrix effects in microbial enumeration tests and their top-down uncertainty evaluation. Microbial counting methods for eighteen pharmaceutical products were validated concerning the trueness (mean recovery) and precision (repeatability and intermediate precision) using seven test microorganisms. Uncertainty factors values were found to be between 1.1 and 3.3, based on trueness and precision results. Trueness uncertainty component was the most relevant in 59% of the cases. This issue can be explained due to the matrix interference cause by preservatives or antimicrobial agents, particularly for lower dilutions when compared to higher dilutions. Thus, we concluded that the higher the matrix interference the higher the uncertainty factor, and consequently, the higher the asymmetry for the interval around the measurement.