Corrugated cardboard boxes are one of the largest paper-based packaging forms used for shipping and handling of wide variety of products in different end-use industries due to low cost, low weight and recyclability. Due to its organic composition, they are highly susceptible to spoilage from heat-resistant microbial spores, leading to economic losses and health risks. In this study, the efficacy of lipopeptides produced from Bacillus amyloliquefaciens MTCC 10456 against thermotolerant Thermoascus crustaceus, Neosartorya hiratsukae and Bacillus subtilis, isolated from spoiled cardboard boxes, was investigated. Lipopeptides were isolated by salt-precipitation of fermentation broth and activity-guided Reverse Phase-High Performance Liquid Chromatography (RP-HPLC). Inhibitory fractions consisted of bacillomycin D and surfactin, which were identified using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS/MS) analysis. Mixture of lipopeptides with nisin (3:2 w/w) asserted significant synergistic effect on the tested pathogens which reduced the minimum inhibitory concentrations (MIC) values and increased their inhibition spectra. Preservative coating containing lipopeptides and nisin was applied on the corrugated cardboard surfaces by mixing with starch-based additive by spread-coating method. It demonstrated biopreservative efficacy against the targeted microorganisms at during the observational period of 180 days. Reduction in microbial count of 4 log cycles was observed in 20 days and showed controlled release of coated peptides which indicate its suitability for packaging purposes. Findings from this study suggests an effective and scalable strategy to prevent microbial spoilage thereby extending the storage period of cardboard boxes.