There is a disagreement on the UV C fluence required for microbial inactivation due to various understanding of UV-C fluence and its measurement methods. This study determined the UV-C fluences required for dairy-associated Listeria monocytogenes strains inactivation using a collimated beam device. In PBS, a UV-C fluence of 10 mJ/cm2 was sufficient to inactivate 5 logs of L. monocytogenes. Milk fat and protein concentration, fat globule and casein micelle particle sizes strongly correlated with observed UV C absorption and scattering of the milk. These optical properties significantly affected the inactivation of L. monocytogenes. Taking into consideration the UV C absorption and reduced scattering coefficients of milk in the UV C fluence calculation, inactivation similar to that observed in PBS at various UV C doses could be achieved in goat and cow milk at various fat levels. This study demonstrated that the inclusion of the milk optical properties is critical for accurate UV C fluence calculations.