Manouri, a protected designation of origin (PDO) cheese, is one of the most popular whey cheeses produced in Greece. The objective of the current study was to investigate if there are differences between artisanal and industrial Manouri cheeses regarding microbiological quality, volatile organic compounds (VOCs) profile and other quality parameters (colour, texture), sensory attributes and spectral characteristics detected by Fourier transform infrared spectroscopy (FT-IR) that may discriminate the samples. Differences were detected in the population of the dominant microbial groups, especially for lactic acid bacteria (LAB), Enterobacteriaceae and yeast counts. No discrimination was attained from the physicochemical analyses, except for the pH values. A total of 50 VOCs were identified, including ketones, lactones, free fatty acids, aldehydes, esters, alcohols and hydrocarbons. Sensory evaluation was carried out using a quantitative descriptive analysis (QDA) panel and a consumer panel. Consumers showed a preference for the artisanal Manouri, and the QDA panel revealed significant differences in 11 out of the 17 sensory attributes. Colour and texture analyses were also performed and showed specific differences in yellowness, as well as in fracturability and hardness. FT-IR spectral analysis demonstrated potential discrimination related to the phospholipid content and profile of artisanal and industrial Manouri.