Purpose: In the current investigation, an ultrasonic approach was performed to produce menadione sodium bisulfite-loaded solid lipid nanoparticles (MSB-SLNs) with rhamnolipid as bio-surfactant which aimed to increase the dermal delivery and anti-pigmentation effect. Method: To achieve optimal skin delivery for MSB, the impact of the ratio of two surfactants (rhamnolipid: Tween) on nanoparticle attributes and the respective functions were evaluated. In vitro diffusion process, in vitro cytotoxicity assay, determination of melanin content of melanoma cells, L-DOPA auto-oxidation inhibitory test, and skin irritation studies carried out for investigation of suitability of MSB formulation in dermal application. Results: The optimized nanoparticles showed an average particle size, zeta potential, polydispersity index, and drug entrapment efficiency of 117.26± 1.12 nm, -6.28± 0.33 mV, 0.262± 0.002, 83.34± 0.75% respectively in hydrophilic-lipophilic balance (HLB) of 12. The in vitro diffusion process demonstrated that MSB-SLN gel had a prolonged release pattern. The levels of MSB in the cutaneous layers (52.192± 2.730% or 961.59± 50.313 μg/cm2) and the receiver compartment (23.721± 1.803 % or 437.049± 33.23665 μg/cm2) for the MSB-SLN gel was higher in compare to MSB simple and showed no cutaneous irritancy and toxicity in rats. MSB-SLN inhibited melanin formation and was remarkably higher than free MSB. MSB-SLN inhibited L-3,4- dihydroxyphenylalanine (L-DOPA) auto-oxidation to a greater extent (95.14± 1.46%) than MSB solution (72.28 ± 0.83%). Conclusion: This study's observations revealed that the produced MSB-SLN might be used as a potential nano-vehicle for MSB dermal administration, thereby opening up innovative options for the management of hyper-melanogenesis problems.