Plasmid conjugation is a key facilitator of horizontal gene transfer. Since plasmids often carry antibiotic resistance genes, they are crucial drivers of the world-wide rise of antibiotic resistance among pathogens. In natural, engineered and clinical environments, bacteria often grow in protective biofilms. Therefore, a better understanding of plasmid transfer in biofilms is needed. Our aim was to investigate plasmid transfer in a biofilm adapted wrinkly colony mutant of Xanthomonas retroflexus (XRw) with enhanced matrix production and reduced motility. We found that XRw biofilms had an increased uptake of the broad host-range IncP-1ϵ plasmid pKJK5 compared to the wild type. Proteomics revealed fewer flagellum associated proteins in XRw, suggesting that flagella were responsible for reducing plasmid uptake. This was confirmed by the higher plasmid uptake of non-flagellated ∆fliM mutants of X. retroflexus wild type and wrinkly mutant. Moreover, testing several flagella mutants of Pseudomonas putida suggested that the flagella effect was more general. We identified seven mechanisms with the potential to explain the flagella effect and simulated them in an individual-based model. Two mechanisms could thus be eliminated (increased distances between cells and increased lag times due to flagella). Another mechanism identified as viable in the modelling was eliminated by further experiments. Four additional proposed mechanisms have a reduced probability of plasmid transfer in common. Our findings highlight the important yet complex effects of flagella during bacterial conjugation in biofilms.