Antimicrobial lipid nanoparticles composed of monoglycerides offer a promising strategy to inhibit membrane-enveloped viral and bacterial pathogens. However, previous efforts mainly focused on fabricating nanoparticles from long-chain monoglycerides, which lack intrinsic antimicrobial activity but contribute to nanoparticle stability and structural integrity. In contrast, shorter-chain monoglycerides often exhibit potent antimicrobial effects but do not self-assemble into colloidally stable nanoparticles and lose efficacy upon dilution. To overcome these limitations and incorporate antimicrobial monoglycerides into a stable nanoparticle configuration, we report a solvent-free microfluidic fabrication strategy that combines the functional characteristics of different monoglycerides to prepare interfacially active, monoglyceride-based nanoparticles with mixed compositions that display potent antibacterial activity. Unlike conventional microfluidic mixing methods that rely on volatile organic solvents, our approach utilizes pharmaceutical-grade materials and does not require organic solvent removal, hence eliminating the need for a dialysis step postfabrication. Dynamic light scattering (DLS) and zeta potential measurements verified that the fabricated nanoparticles had ∼250-350 nm diameters and exhibited high colloidal stability whereas the antibacterial activity of the nanoparticles against