We established a protocol for the biochemical fractionation of Agrobacterium tumefaciens membranes, and characterized the proteome of detergent-sensitive and detergent-resistant membrane fractions by mass spectrometry. We found that well-established microdomain marker proteins co-purify with proteins associated with transport or secretion processes in the detergent-resistant fraction, suggesting a functional micro-compartmentalization of the Gram-negative membrane. The presence of components of virulence-associated secretion systems in detergent-resistant membranes supports the involvement of bacterial microdomains in host-microbe interaction and microbial competition.