Application of hyperspectral imaging (HSI) and data analysis algorithms was investigated for early and non-destructive detection ofBotrytis cinereainfection. Hyperspectral images were collected from laboratory-based contaminated and non-contaminated fruits at different day intervals. The spectral wavelengths of 450 nm to 900 nm were pretreated by applying moving window smoothing (MWS), standard normal variates (SNV), multiplicative scatter correction (MSC), Savitzky–Golay 1stderivative, and Savitzky–Golay 2ndderivative algorithms. In addition, three different wavelength selection algorithms, namely; competitive adaptive reweighted sampling (CARS), uninformative variable elimination (UVE), and successive projection algorithm (SPA), were executed on the spectra to invoke the most informative wavelengths. The linear discriminant analysis (LDA), developed with SNV-filtered spectral data, was the most accurate classifier to differentiate the contaminated and non-contaminated kiwifruits with accuracies of 96.67% and 96.00% in the cross-validation and evaluation stages, respectively. The system was able to detect infected samples before the appearance of disease symptoms. Results also showed that the gray-mold infection significantly influenced the kiwifruits’ firmness, soluble solid content (SSC), and titratable acidity (TA) attributes. Moreover, the Savitzky–Golay 1stderivative-CARS-PLSR model obtained the highest prediction rate for kiwifruit firmness, SSC, and TA with the determination coefficient (R2) values of 0.9879, 0.9644, 0.9797, respectively, in calibration stage. The corresponding cross-validation R2values were equal to 0.9722, 0.9317, 0.9500 for firmness, SSC, and TA, respectively. HSI and chemometric analysis demonstrated a high potential for rapid and non-destructive assessments of fungal-infected kiwifruits during storage.