Bio-deterioration of buildings is a big challenging issue worldwide. Therefore, the current study quested for finding ecofriendly approach against building bio-deteriorator microbes. Chitosan nanoparticles (CNPs) are natural polymeric nanoparticles with unique characteristics and exhibits potent antimicrobial activities, making them a promising eco-friendly material for biocontrol. Thus, this study describes an alternative, green synthesis approach for CNPs using the cell free supernatant of Paradendryphiella sp. as a crosslinking and stabilizing agent. The SEM and TEM analyses showed that CNPs are spherical particles, with sizes ranging from 10.1 to 26.08 nm. The presence of several bands in the FTIR spectrum shows the existence of capping agents that stabilize CNPs. X-ray diffraction reveals the crystalline structure of CNPs. A time-dependent antifungal assay of CNPs was implemented against Exophiala dermatitidis as a model of slow growing meristematic black fungi that causes building biodeterioration. The results revealed that upon employing the higher doses of CNPs (250 and 500 µg/mL), significant fungicidal potency was exhibited within 33 and 57 h exposure time, with significant (P–value ˂ 0.0001) lethality rate (K-value) of -0.06 and -0.009 CFU/ml/h, respectively. Further, SEM micrographs illustrated the prevalence of morphologically deteriorated macroconidia or budding yeast cells without polymeric matrix of biofilm with minor presence of crinkled mycelia and avoiding of pseudo-hyphae and sclerotic bodies, reflecting thereby the losing of cells pathogenicity through lethal antimycotic potentiality of CNPs. Hence, the current study highlights the possibility of employing the mycogenically fabricated CNPs as hygienic coating in maintaining building structures from bioreceptivity and biodeterioration.