Background: Highly active antiretroviral therapy (HAART) does not allow the complete clearance of the virus since it does not target viral reservoirs nor efficiently block HIV-1 cell-to-cell transmission in vivo. HIV-1 cell-to-cell spread is thousands-fold more efficient than cell-free infection; yet, how virions are transferred via cell contacts remains unknown. Methods: Using a panel of cutting-edge imaging techniques (Cryo- TEM, CL/SEM, CL/FIB and super-resolution imaging) to functional assays, we investigated and characterized viral structures involved in HIV-1 cell-associated infectivity. We analysed a range of infected T-cell cultures (chronically infected T-cell lines, primary CD4+ T cells infected in vitro with virus primary isolates and CD4+ T lympho- cytes from untreated HIV-1-infected patients with a detectable viraemia). Results: We show here that HIV-1 cell-associated infectivity mostly resides at the surface of CD4+ T lymphocytes in a viral biofilm, formed by viral particles aggregated within a scaffold ofextracellular matrix (ECM) components. Our set of data demon- strates that (i) biofilm-associated viral particles are more effi- cient in establishing infection than free viral particles and (ii) they confer HIV with important properties characterizing cell-to- cell spread. This includes the decreased sensitivity to HAART and to the broad neutralizing antibody 3BNC117. HIV-1 regu- lates biofilm matrix composition that controls both viral parti- cles organization at the cell surface and the resulting cell- associated infectivity. The organized clustering of viral particles along an ECM framework locally concentrates virions, favours their collective transfer to target cells and limits their exposure to nAbs. Finally, CD4+ T cells from HIV-1-infected patients pro- duce and transmit viral biofilms, supporting that they may also be involved in vivo. Conclusions: This study thus unveils HIV biofilm as a new highly infectious extracellular entity that concentrates, stores, dissemi- nates and shelters HIV-1 infectivity. This may have implications for HIV-1 spread and persistence in host, including for maintain- ing HIV-1 sanctuaries in treated patients. Our results unveil a new role for the ECM in clustering and protecting HIV-1 at the plasma membrane and in their collective transfer through viro- logical synapses. Targeting biofilm ECM components could repre- sent a promising approach to favour HIV-1 clearance or to potentiate the effect of available anti-viral therapies.