Melaleuca alternifolia tea tree oil (TTO) is largely used in cutaneous infections. Clinical observations reported antibacterial, antifungal, and antiviral activities, whereas in vitro experiments ascribed most of biological properties to terpinen-4-ol. Since different plant chemotypes and storage conditions result in variations of chemical composition of commercially available TTO, in this study we investigated the antimicrobial activity and the chemical profile of ten commercially available TTO products. The antimicrobial activity was assessed against Candida glabrata, Herpes simplex virus type 1 (HSV-1), methicillin-resistant Staphylococcus aureus (MRSA), and Pseudomonas aeruginosa grown in planktonic mode or biofilms. Only five out of ten TTO batches reported significant antimicrobial activity. The identified TTO products reduced bacterial survival in biofilms, generated oxidative damage in C. glabrata, and diminished HSV-1 infectivity. GC-MS analysis revealed that all the analyzed TTO batches fitted into the terpinen-4-ol chemotype even if we reported great variability in composition of nine major ISO-specified TTO components. Overall, we were not able to ascribe the antimicrobial activity to the content in terpinen-4-ol. We therefore conclude that the antimicrobial activity of TTO results from complex interaction among different components.