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Abstract A new technique was used to measure the viscoelasticity of in vivoPseudomonas aeruginosabiofilms. This was done through ex vivo microrheology measurements of in vivo biofilms excised from mouse wound beds. To our knowledge, this is the first time that the mechanics of in vivo biofilms have been measured. In vivo results are then compared to typical in vitro measurements. Biofilms grown in vivo are more relatively elastic than those grown in a wound-like medium in vitro but exhibited similar compliance. Using various genetically mutatedP. aeruginosastrains, it is observed that the contributions of the exopolysaccharides Pel, Psl, and alginate to biofilm viscoelasticity were different for the biofilms grown in vitro and in vivo. In vitro experiments with collagen containing medium suggest this likely arises from the incorporation of host material, most notably collagen, into the matrix of the biofilm when it is grown in vivo. Taken together with earlier studies that examined the in vitro effects of collagen on mechanical properties, we conclude that collagen may, in some cases, be the dominant contributor to biofilm viscoelasticity in vivo.