Coffee is one of the most widely consumed beverages worldwide due to its sensory and potential health-related properties. In the present comparative study, a preparation known as Greek or Turkish coffee, made with different types/varieties of coffee, has been investigated for its physicochemical attributes (i.e., color), antioxidant/antiradical properties, phytochemical profile, and potential biological activities by combining high-throughput analytical techniques, such as infrared spectroscopy (ATR-FTIR), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and in silico methodologies. The results of the current study revealed that roasting degree emerged as the most critical factor affecting these parameters. In particular, the L* color parameter and total phenolic content were higher in light-roasted coffees, while decaffeinated coffees contained more phenolics. The ATR-FTIR pinpointed caffeine, chlorogenic acid, diterpenes, and quinic esters as characteristic compounds in the studied coffees, while the LC-MS/MS analysis elucidated various tentative phytochemicals (i.e., phenolic acids, diterpenes, hydroxycinnamate, and fatty acids derivatives). Among them, chlorogenic and coumaric acids showed promising activity against human acetylcholinesterase and alpha-glucosidase enzymes based on molecular docking studies. Therefore, the outcomes of the current study provide a comprehensive overview of this kind of coffee preparation in terms of color parameters, antioxidant, antiradical and phytochemical profiling, as well as its putative bioactivity.