<p>Foodborne pathogens remain a major public health concern, necessitating reliable and rapid detection methods suitable for diverse food matrices. This study evaluated culture-based ISO methods, a molecular LAMP-based system (3&#xa0;M™ Molecular Detection System, 3&#xa0;M MDS), and an immunoassay-based platform (VIDAS® ELFA) for the detection of <i>Salmonella</i> spp., <i>Listeria monocytogenes</i> (alone and in the presence of <i>Listeria innocua</i>), <i>Escherichia coli</i> O157:H7, and <i>Campylobacter</i> spp. in a controlled UHT milk matrix. Samples were artificially contaminated under low-level (10¹–10²&#xa0;CFU/mL), high-level (10⁴–10⁵&#xa0;CFU/mL), and heat-inactivated conditions. At high contamination levels, all methods consistently detected most target pathogens. Under low-level contamination, method-dependent variability was observed, particularly for <i>E. coli</i> O157:H7 and mixed <i>Listeria</i> populations. Variable molecular detection of <i>E. coli</i> O157:H7 may be associated with instability of <i>stx</i> genes, whereas the chromosomally encoded <i>eae</i> gene remained consistently detectable. In samples containing both <i>L. monocytogenes</i> and <i>L. innocua</i>, recovery of <i>L. monocytogenes</i> was inconsistent across methods, indicating competitive effects during enrichment. In heat-inactivated samples, direct plating confirmed loss of culturability following thermal treatment; however, positive results were occasionally detected, possibly due to residual DNA or antigenic components. This study provides a harmonized comparison of ISO, 3&#xa0;M MDS, and VIDAS across multiple foodborne pathogens under both viable and heat-inactivated conditions, addressing less explored aspects. Overall, the findings provide biological context for method-dependent detection outcomes and highlight the complementary roles of culture-based and rapid detection approaches, emphasizing context-aware interpretation in routine testing.</p> Graphical Abstract <p></p>