Background: Antimicrobial peptides (AMPs) are short, cationic, amphipathic molecules that have gained tremendous popularity as alternatives to traditional antibiotics due to their lower propensity to develop bacterial resistance. However, the clinical developability of AMPs remains impeded due to shortcomings such as proteolytic instability and poor penetration leading to low bioavailability. Aims: To improve the access of AMPs to cells and subsequent bacteria killing, we evaluated the cell-penetrating and antimicrobial properties of three novel libraries of synthetic peptoids using Minimum Inhibitory Concentration, killing efficacy and membrane permeabilization assays against mycobacteria and Staphylococcus aureus. In addition, we investigated cell selectivity using mammalian cells to assess peptoid toxicity. Results: We showed that short tetrameric Rhodamine B-labeled peptoids composed of a balance of aromatic and lipophilic residues have potent selective antimicrobial activity against a range of microorganisms. The most potent candidates were active against drug-resistant S. aureus isolates as well as mycobacterial strains, with cell penetrating capabilities reported in HeLa and RAW 264.7 macrophage cells. Conclusions: These data suggest that peptoids with novel dual functionalities may potentially be an interesting class of therapeutics and/or molecular delivery agents for anti-infective purposes.