The emergence of resistance to clinically used antibiotics by bacteria presents a significant problem in public health. Natural antimicrobial peptides (AMPs) are a valuable source of antibiotics that act by a mechanism less prone to the evolutionary development of resistance. In an effort to realize the promise of AMPs while overcoming limitations such as poor biostability, researchers have sought sequence-defined oligomers with artificial amide-based backbones that show membrane-disrupting functions similar to natural agents. Most of this precedent has focused on short peptidomimetic analogues of unstructured chains or secondary folds; however, the natural antimicrobial arsenal includes a number of small- and medium-sized proteins that act via an ordered tertiary structure. Generating proteomimetic analogues of these scaffolds poses a challenge due to the increased complexity of the target for mimicry. Here, we report the development of heterogeneous-backbone variants of lasiocepsin, a 27-residue disulfide-rich AMP found in bee venom that adopts a compact tertiary fold. Iterative cycles of design, synthesis, and biological evaluation yielded analogues of the natural domain with ∼30 to 40% artificial backbone content, comparable antibacterial activity, reduced host cell toxicity, and improved stability to proteolytic degradation. High-resolution structures determined for several variants by NMR provide insights into the interplay among backbone composition, tertiary fold, and biological properties. Collectively, the results reported here broaden the scope of protein functional mimicry by artificial backbone analogues of tertiary folding patterns and suggest protein backbone engineering as a means to tune protein function by exerting site-specific control over protein folded structure.