Conjugal plasmid transfer is an efficient mechanism for gene exchange among bacteria. Most bacteria exist in biofilms encased in extracellular polymeric substances (EPS), which provide protection against environmental stressors such as water deprivation. We hypothesized that enhanced EPS production in response to water matric stress would create a physical barrier limiting exogenous plasmid invasion into established biofilms. Employing filter mating assays, we demonstrate that Pseudomonas putida (serving as recipient strain), which produces more EPS with decreasing water matric potential, suppresses plasmid invasion from exogenously added P. putida (pKJK5) donor cells. Similarly, transfer into a biofilm formed by an EPS overproducing P. putida mutant was impaired. This barrier effect was not observed in biofilms co-established by mixtures of donor and recipient strains, probably because EPS does not form a thick enough internal barrier within the biofilm compared to the external barrier on top of a mature biofilm. Hence, sufficiently high cell-to-cell contacts remain possible within these biofilms regardless of water matric stress and EPS production capability. We further tested these mechanisms employing a complex, natural soil bacterial community as recipient; also here conjugal plasmid invasion declined with decreasing matric potential. Our study provides novel insight into the complex dynamics of horizontal transfer of plasmids in microbial biofilms.