Although lentiviral transduction methods are widely used, their broader application is dependent upon the optimization of lentiviral transduction efficiency for a broad range of cell types. In the present study, we focus on the evaluation of two chemical classes with respect to their ability to increase lentiviral transduction without cytotoxicity. We compared the activity of adjuvants that are already used for lentivirus delivery with that of novel adjuvants selected on the basis of their chemical and physical characteristics. The novel poloxamer synperonic F108 demonstrated superior characteristics for enhancing lentiviral transduction over the best-in-class polybrene-assisted transduction. The results revealed that poloxamer synperonic F108 exhibited the dual benefits of low toxicity and a high efficiency of lentiviral gene delivery into a range of different primary cell cultures. In the presence of poloxamer synperonic F108, cells showed an increased propidium dye influx indicating a re-organization of membrane microstructures accompanying lentivirus uptake. The administration of a mixture of poloxamer synperonic F108 with polybrene further enhanced lentiviral transduction rates. The results obtained in the present study indicate that a contribution to efficiency is made by each adjuvant, with polybrene acting as a charge protector and poloxamer synperonic F108 as a membrane modulator. Therefore, poloxamer synperonic F108, either alone or in combination, can lead to the optimization of large-scale lentiviral transduction approaches.