Thymoquinone (TQ), a pleiotropic compound isolated from the seeds of Nigella sativa has a great potential as a chemotherapeutic agent against several cancers. However, its limited aqueous solubility and poor stability impede its clinical utility. To overcome these hurdles, TQ was encapsulated into spanlastics made using Span 60 and various edge activators. The spanlastics were evaluated for particle size, polydispersity index, zeta potential, drug entrapment efficiency and in vitro drug release. TQ anticancer efficacy was tested in vitro against breast cancer cell line MCF-7. TQ-loaded spanlastics had spherical shape, particle size in the range of 92–285 nm and negative zeta potential (around −12 to −25 mV). The particle size and zeta potential were greatly influenced by the type and concentration of used edge activator. Tween 80 spanlastics had the smallest particle size (around 90–110 nm). High drug entrapment efficiency was observed for all the tested edge activators (around 76–99 %) and it was possible to modulate it by varying the edge activator and drug concentrations. Drug release rate was also dependent on the type and concentration of the edge activator. Tween 80 spanlastics, used as an optimum formulation resulted in 11.5- and 5-fold increase in TQ cytotoxic efficacy against MCF-7 cells compared with the free drug and the drug loaded into conventional niosomes, respectively. These results confirm that Tween 80 spanlastics could be a promising nano-delivery system to enhance the anticancer efficacy of TQ or similar anticancer drugs.