Coffee consumption decreases the risk of oxidative stress-related diseases. The by-product obtained after brewing process (spent coffee) also has antioxidant capacity. Spent coffee and coffee brews (filter and espresso) extracts were obtained from Arabica and Robusta coffees, respectively. Spent coffee showed slightly high amounts in chlorogenic acids, but caffeine content was similar to their respective coffee brew. All samples exhibited strong protection activity against indirect acting mutagen 2-AF (≤92%), whereas the protection against NPD (direct mutagen) was 12–35% (Ames Test). The growth inhibition of common food-borne pathogen and food spoilage microorganisms by coffee extracts was also studied. Spent coffee showed antimicrobial activity, mainly against Gram-positive bacteria (Staphylococcus aureus, Listeria monocytogenes) and yeast (Candida albicans). The role of phenolic acids, caffeine and melanoidins in the antimutagenic and antimicrobial activities is discussed. Thus, spent coffee extracts could be a potential source of bioactive compounds, thereby becoming a promising new functional food ingredient