Biofilms can constitute permanent threats to food safety and public health. Bacteria and viruses lodged in biofilm can escape cleaning and sanitizing agents. The aim of this study was to compare Pseudomonas aeruginosa developing and mature biofilms produced on agri-food surfaces in terms of interaction with murine norovirus. Whether they were mature or still developing the biofilms apparently accumulated murine norovirus in large numbers after 24 h of contact with medium which viral titer was 2.6 × 10⁴ pfu ml⁻¹ (≈ 8 log₁₀ genome copies ml⁻¹). This appeared unrelated to surfaces’ nature and bacterial viable count but related to polysaccharide and protein contents. Virus releases may also occur mainly in connection with P. aeruginosa biofilm dispersal systems. These findings suggest that the effectiveness of surface cleaning agents and procedures for reducing the risks of biofilms-related viral contaminations need to be re-evaluated in relation with biofilm components. However, more repetitions and further in-depth specific studies are needed for confirmation of these findings and more clarifications on virus-biofilm interaction phenomenon.