Clinical limitations and drug-resistance are the basis of the search for new antifungal therapeutics against biofilm forming pathogen Aspergillus fumigatus. Cis-9-hexadecenal is a natural compound present in various plants extracts, but its antifungal activity against A. fumigatus is still unexplored. The aim of present study was to evaluate the antifungal efficacy of cis-9-hexadecenal on A. fumigatus, specifically its biofilm and to assess its cytotoxicity. Broth micro-dilution method revealed that cis-9-hexadecenal inhibited 90% of planktonic growth of A. fumigatus at 0.078 mg/ml. In-vitro combinatorial effect between drug amphotericin B and cis-9-hexadecenal was studied by checkerboard assay. Cis-9-hexadecenal, in combination to amphotericin-B had additive effect and showed enhanced drug efficacy against A. fumigatus. The effect of cis-9-hexadecenal on pre-formed biofilm was analyzed through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and scanning electron microscopy. In MTT assay, minimum biofilm-eradicating concentration-80 of cis-9-hexadecenal was 0.156 mg/ml. Scanning electron micrograph showed absence of extracellular matrix and tangled hyphae in cis-9-hexadecenal treated biofilm whereas these structures were present in both untreated and amphotericin-B treated biofilm. Drug-likeness was predicted through in-silico ADMET studies. The study envisaged that cis-9-hexadecenal followed Lipinski’s rule but had a higher Log P value indicating that it’s not hydrophilic. The compound can be modified to enhance the absorption and permeation. Cell cytotoxicity study exhibited that cis-9-hexadecenal was not toxic to normal human lung epithelial cell line L-132 upto 0.62 mg/ml. The study concluded that cis-9-hexadecenal might be explored further as a potential therapeutic molecule for A. fumigatus associated diseases.