Staphylococcal enterotoxin B (SEB), encoded by the seb gene, is a virulence factor produced by Staphylococcus aureus that is involved mainly in food poisoning and is known to act as an aggravating factor in patients with atopic dermatitis. Research results in animal infection models support the concept that superantigens, including SEB contribute to sepsis and skin and soft tissue infections. In contrast to antibiotics, antimicrobial photodynamic inactivation (aPDI) is a promising method to combat both bacterial cells and virulence factors. The main aims of this research were to (1) select the most stable reference genes under sublethal aPDI treatments and (2) evaluate the impact of aPDI on seb. Two aPDI combinations were applied under sublethal conditions: rose bengal (RB) and green light (λmax = 515 nm) and new methylene blue (NMB) and red light (λmax = 632 nm). The stability of ten candidate reference genes (16S rRNA, fabD, ftsZ, gmk, gyrB, proC, pyk, rho, rpoB and tpiA) was evaluated upon aPDI using four software packages-BestKeeper, geNorm, NormFinder and RefFinder. Statistical analyses ranked ftsZ and gmk (RB + green light) and ftsZ, proC, and fabD (NMB + red light) as the most stable reference genes upon photodynamic treatment. Our studies showed downregulation of seb under both aPDI conditions, suggesting that aPDI could decrease the level of virulence factors.