We present the synthesis and the in vitro assaying of a series of lactose-based non-ionic surfactants, highlighting the relationship between their structure and biological effect. Using tensiometric measurements the critical micelle concentrations (CMCs) of the surfactants were determined and demonstrate that increasing hydrophobic chain length reduces surfactant CMC. In vitro testing on Caco-2 intestinal and Calu-3 airway epithelia revealed that cytotoxicity is present, for most of the surfactants, at concentrations greater than their CMCs. Importantly, through the culture of epithelial monolayers on Transwell® supports, the surfactants demonstrate the ability to reversibly modulate transepithelial electrical resistance (TEER), and thus open tight junctions, at non-toxic concentrations. The surfactants were then tested for their ability to improve the in vitro permeability of Ovalbumin and Dextran, confirming their potential application as safe permeability enhancers in vivo. Moreover, the synthesized compounds exhibit antimicrobial activity versus eight pathogenic species belonging to Gram-positive, Gram-negative microorganisms and fungi.