Swarming motility and biofilm formation are opposite, but related surface-associated behaviors that allow various pathogenic bacteria to colonize and invade their hosts. In Sinorhizobium meliloti, the alfalfa endosymbiont, these bacterial processes and their relevance for host plant colonization are largely unexplored. Our previous work demonstrated distinct swarming abilities in two S. meliloti strains (Rm1021 and GR4) and revealed that both environmental cues (iron concentration) and bacterial genes (fadD, rhb, rirA) play crucial roles in the control of surface motility in this rhizobial species. In the current study, we investigate whether these factors have an impact on the ability of S. meliloti to establish biofilms and to colonize host roots.We found that strain GR4, which is less prone to translocate on solid surfaces than strain Rm1021, is more efficient in developing biofilms on glass and plant root surfaces. High iron conditions, known to prevent surface motility in a wild-type strain of S. meliloti, promote biofilm development in Rm1021 and GR4 strains by inducing the formation of more structured and thicker biofilms than those formed under low iron levels. Moreover, three different S. meliloti mutants (fadD, rhb, and rirA) that exhibit an altered surface translocation behavior compared with the wild-type strain, establish reduced biofilms on both glass and alfalfa root surfaces. Iron-rich conditions neither rescue the defect in biofilm formation shown by the rhb mutant, which is unable to produce the siderophore rhizobactin 1021 (Rhb1021), nor have any impact on biofilms formed by the iron-response regulator rirA mutant. On the other hand, S. meliloti FadD loss-of-function mutants do not establish normal biofilms irrespective of iron levels.Our studies show that siderophore Rhb1021 is not only required for surface translocation, but also for biofilm formation on glass and root surfaces by strain Rm1021. In addition, we present evidence for the existence of control mechanisms that inversely regulate swarming and biofilm formation in S. meliloti, and that contribute to efficient plant root colonization. One of these mechanisms involves iron levels and the iron global regulator RirA. The other mechanism involves the participation of the fatty acid metabolism-related enzyme FadD.