Abstract <p>Whey permeate (WP), the main waste stream of the dairy industry, was used as a raw material to produce fully bio-based non-ionic surfactants. Specifically, on the one hand, WP was submitted to a transglycosylation reaction catalyzed by the immobilized β-galactosidase from <i>Aspergillus oryzae</i> in 1-BuOH, affording 1-butyl β-D-galactopyranoside (yield 40%), which was used as the polar “head” of the surfactant. On the other hand, a WP-based fermentation process by the yeast <i>Cutaneotrichosporon oleaginosus</i> ATCC 20509 was employed to produce single cell oil (45% w/w_{cell dry weight}). The microbial lipids were recovered from the freeze-dried cells and derivatized in a one-pot system by acid-catalysis to yield the corresponding ethyl esters as apolar “tails” (75% w/w yield, based on lipid content). These bio-based building blocks were converted into the sugar fatty acid esters (SFAE) <i>n</i>-butyl 6-<i>O</i>-acyl-β-D-galactopyranosides by a lipase-catalyzed transesterification reaction (yield 40%). The hydrophilic–lipophilic balance and solubility parameters of the synthesized SFAE mixture were calculated. Additionally, its ability to reduce the interfacial tension was examined, including the effect of fatty tail unsaturation. The interfacial performance of the SFAE mixture, containing both palmitic (45%) and oleic (40%) acid residues, was lower (6.3 mN m⁻¹) compared to the SFAE containing only palmitic acid as the fatty acid tail (0.1 mN m⁻¹) at the same concentration, but still highly promising.</p> Key points <p>• <i>Whey permeate (WP) is the main waste stream of dairy industry.</i></p> <p>• <i>WP was upcycled by coupling fermentation and biocatalysis.</i></p> <p>• <i>Bio-based surfactants (sugar fatty acid esters) were obtained using WP as biomass.</i></p> Graphical Abstract <p></p>