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Se nanoparticles (NPs) of predominantly amorphous phase (α-Se) have been prepared in room-temperature ionic liquids (RTILs). The effects of ion-pair combination and −OH functionalization of RTILs on the size and phase stability of Se NPs were investigated. The RTILs used were 1-ethyl-3-methyl imidazolium boron tetrafluoride ([EMIM]­[BF4]), 1-(2-hydroxyethyl)-3-methyl imidazolium boron tetrafluoride ([EOHMIM]­[BF4]), and 1-ethyl-3-methyl imidazolium methane sulfonate ([EMIM]­[MS]). The size of Se NPs@[EOHMIM]­[BF4] was found to be the smallest (∼32 nm), followed by Se NPs@[EMIM]­[BF4] (∼57 nm) and Se NPs@[EMIM]­[MS] (∼60 nm), respectively. Interestingly, the stability studies revealed minimal size variations for Se NPs@[EMIM]­[MS], followed by Se NPs@[EOHMIM]­[BF4] and Se NPs@[EMIM]­[BF4], respectively. The observed trends could be correlated with the strength of interionic interactions in the respective RTILs, as well as their packing order (density). Importantly, the RTILs played the role of a solvent, a stabilizer, and an in situ source of reducing species. Pulse radiolysis study revealed imidazolium-originated radical species-driven formation of Se NPs. Further, anticancer efficacy studies demonstrated the role of NP size, wherein Se NPs@[EOHMIM]­[BF4] exhibited the highest cancer cell killing, followed by Se NPs@[EMIM]­[BF4] and Se NPs@[EMIM]­[MS]. Another significant highlight of this work is the reuse of the spent RTILs for the synthesis of the next batch of Se NPs.