<jats:title>Abstract</jats:title><jats:p>The viscosin group covers a series of cyclic lipodepsipeptides (CLPs) produced by <jats:italic>Pseudomonas</jats:italic> bacteria, with a range of biological functions and antimicrobial activities. Their oligopeptide moieties are composed of both <jats:sc>L</jats:sc>‐ and <jats:sc>D</jats:sc>‐amino acids. Remarkably, the Leu5 amino acid—centrally located in the nonapeptide sequence—is the sole residue found to possess either an <jats:sc>L</jats:sc> or <jats:sc>D</jats:sc> configuration, depending on the producing strain. The impact of this <jats:sc>D</jats:sc>/<jats:sc>L</jats:sc> switch on the solution conformation was investigated by NMR‐restrained molecular modelling of the epimers pseudodesmin A and viscosinamide A. Although the backbone fold remained unaffected, the <jats:sc>D</jats:sc>/<jats:sc>L</jats:sc> switch adjusted the segregation between hydrophobic and hydrophilic residues, and thus the amphipathicity. It also influenced the self‐assembly capacity in organic solvents. Additionally, several new minor variants of viscosinamide A from <jats:italic>Pseudomonas fluorescens</jats:italic> DR54 were identified, and an NMR assay is proposed to assess the presence of either an <jats:sc>L</jats:sc>‐ or <jats:sc>D</jats:sc>‐Leu5.