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The research aim was to determine the effectiveness of disinfection methods: thyme essential oil microatmosphere (TEO), silver nanoparticles misting (AgNPs), and low temperature plasma (LTP) by culture-dependent method and RNA analysis. In addition, we examined the influence of disinfection on mechanical and optical properties of paper from historical books with different levels of microbial contamination. All disinfection methods were generally bacteriostatic and fungistatic. The AgNPs misting method was more effective for bacterial inhibition ( R = 60–100%), than LTP ( R = 25–100%) and TEO ( R = 12–100%); all tested methods were less effective for fungi ( R = 0–99.8%). TEO exhibits a broader spectrum of fungicidal activity compared to AgNPs and LTP. The antimicrobial effectiveness depended on the area from where the microorganisms were isolated and their sensitivity to the active agent. A higher disinfection effectiveness was observed for books with higher levels of microbial contamination. RNA concentration was a good marker of antimicrobial activity of disinfection. RNA amount decreased by 95–100% after disinfection with LTP and TEO, and by 29–89% after AgNPs disinfection. Disinfection of paper from historical books with LTP, AgNPs and TEO did not significantly influence or have a positive effect on the mechanical and optical properties of paper from tested historical books. We show that LTP, TEO, and AgNPs can be used as microbiostatics, alternatively to the currently available methods. [ABSTRACT FROM AUTHOR]