The unique segregation and self-assembly properties of fluorinated amphiphiles into supramolecular architectures have attracted substantial attention in recent years. In this study, we report the design and synthesis of six newer hybrid nonionic amphiphiles with hydrophobic alkyl/fluoroalkyl chains and hydrophilic polyethylene glycol (PEG) units of different lengths and evaluate and compare their drug delivery applications. On the hydrophobic side, the amphiphiles differ in having either two alkyl chains, two fluoroalkyl chains, or one alkyl and one fluoroalkyl chain. This allowed for a systematic comparison of the physicochemical and transport abilities. The aggregation phenomenon of the amphiphiles was monitored with Cryo-TEM. Cytotoxicity and cellular uptake studies were performed to evaluate the pharmacological potential. Candida antarctica lipase (Novozym-435) immobilized on solid support was used to facilitate the release of the encapsulated cargo. Our study highlights the potential and properties of the fluoroalkyl-based nanocarriers.