In vitro culture of any plant tissue requires stringent aseptic conditions to thrive within a microorganism-free environment. This is particularly difficult since culture media inherently contain an optimal and sufficient nutrient concentration for numerous microorganisms, thereby impeding the correct development of the explants. In this study, we assessed the antimicrobial and antifungal efficacy of silver nanoparticles (AgNPs) and the chemical inhibitor of contamination, 1-phenyl-1- (N-phenyl-N’-ethyl-guanil)-3-ethyl-thiocarbamide (Plant Tissue Culture Contamination Control, PTC3) during the propagation of “mashua” MAC003 morphotype (Tropaeolum tuberosum Ruiz & Pavón). We successfully demonstrated the use of these compounds in the medium obviating the need for an autoclaving procedure. To achieve this, we employed a gradient of concentrations of both agents. Murashige and Skoog (MS) culture medium with 3% sucrose and 0.7% agar at pH 5.6 was supplemented with AgNPs nanoparticles at 20, 50, and 100 ppm, alongside PTC3 at 0.2, 2 and 3 ppm concentrations. Both AgNPs and PTC3 exhibited inhibitory effects on microbial and fungal growth across all tested concentrations. Statistical analysis of the biometric parameters measured in explants over 4 week period indicated that the optimal AgNPs concentration was 20 ppm. Additionally, the growth and development outcome of Tropaeolum tuberosum explants were most favorable with PTC3 concentration of 0.2 ppm, as discerned through a comparative analysis of the two compounds. This study proposes the use of PTC3 as a novel compound of choice for avoiding the autoclaving process within in vitro plant tissue culture techniques.