Electrospun nanofibers have improved physicochemical properties compared to macroscalar fibers and are therefore increasingly investigated for use in novel food packaging systems. The objectives of this study were to electrospin nanofibers and to evaluate the effect and feasibility of surfactants and surfactant aggregates to modulate properties and functionalities of electrospun nanofibers. Nanofibers were fabricated by electrospinning a mixture of cationic chitosan and noncharged poly(ethylene oxide) (PEO) in aqueous acetic acid. To improve spinnability and nanofiber morphologies, surfactants were added to biopolymer/polymer solutions and their influence was investigated. Solution properties were evaluated by rheological, surface tension, and conductivity measurements. Fibers were characterized by scanning and transmission electron microscopy, differential scanning calorimetry, and Fourier transform infrared spectroscopy. Addition of PEO and surfactants induced spinnability producing larger fibers with diameters ranging from 40 to 240 nm, while pure chitosan did not form fibers and was instead deposited as beads. Compositional analysis suggested that nanofibers consisted of all solution constituents with chitosan concentrations being significantly lower in fibers than in solution, indicating that surfactants may have decreased polymer-polymer interactions responsible for entanglement. Poly(vinyl alcohol) nanofibers were used as novel delivery system for eugenol carrying Surfynol®465 micelles (microemulsion). Solution properties were not significantly altered after addition of microemulsion regardless of surfactant and/or eugenol concentration. Transmission electron imaging revealed a homogeneous distribution of microemulsion throughout the nanofibers. Release studies suggested a burst release mechanism of encapsulated eugenol microemulsion, potentially due to hydrophilicity of the polymeric carrier, while faster release was observed in samples with a higher eugenol loading ratio in the microemulsion. Antimicrobial activity of produced nanofibers carrying phytophenol microemulsions was evaluated against two strains of Salmonella Typhimurium and Listeria monocytogenes. Overall, the functionalized nanofibers had higher antimicrobial efficacies against Gram-negative than Gram-positive bacterial strains and were also more effective than pure eugenol microemulsion added at respective concentrations to the test system possibly due to a faster exhaustion and loss of antimicrobial activity in free microemulsions. Results of this study suggest that composite solutions of biopolymers, synthetic polymers, and micellar surfactant solutions can be successfully electrospun potentially offering a new means to functionalize biopolymeric nanofibers.