The scientific rationale for selection of the surfactant type during oral formulation development requires an in-depth understanding of the interplay between surfactant characteristics and biopharmaceutical factors. Currently, however, there is a lack of comprehensive knowledge of how surfactant properties, such as hydrophilic-lipophilic balance (HLB), digestibility, and fatty acid (FA) chain length, translate into in vivo performance. In the present study, the relationship between surfactant properties, in vitro characteristics, and in vivo bioavailability was systematically evaluated. An in vitro lipolysis model was used to study the digestibility of a variety of nonionic surfactants. Eight surfactants and one surfactant mixture were selected for further analysis using the model poorly water-soluble drug nilotinib. In vitro lipolysis of all nilotinib formulations was performed, followed by an in vivo pharmacokinetic evaluation in rats. The in vitro lipolysis studies showed that medium-chain FA-based surfactants were more readily digested compared to long-chain surfactants. The in vivo study demonstrated that a Tween 20 formulation significantly enhanced the absolute bioavailability of nilotinib up to 5.2-fold relative to an aqueous suspension. In general, surfactants that were highly digestible in vitro tended to display higher bioavailability of nilotinib in vivo. The bioavailability may additionally be related to the FA chain length of digestible surfactants with an improved exposure in the case of medium-chain FA-based surfactants. There was no apparent relationship between the HLB value of surfactants and the in vivo bioavailability of nilotinib. The impact of this study's findings suggests that when designing surfactant-based formulations to enhance oral bioavailability of the poorly water-soluble drug nilotinib, highly digestible, medium chain-based surfactants are preferred. Additionally, for low-permeability drugs such as nilotinib, which is subject to efflux by intestinal P-glycoprotein, the biopharmaceutical effects of surfactants merit further consideration.