Environmental conditions, including irrigation water quality, are known to affect secondary metabolite production in plants and hence the composition and activity of essential oils of aromatic plants. The aim of this study is to evaluate the effect of irrigation with secondary-treated effluent, as compared with potable water irrigation, on the composition, biological activities and yield of essential oil (EO) from Origanum syriacum L. var. syriacum (OSS). The EO from OSS cultivated in Palestine under secondary treated municipal effluent compared with potable water irrigation were analyzed seasonally over a two-year period for oil quantity and composition, antioxidant activities 1,1- diphenyl-2-picrylhydrazyl (DPPH), 2,2 azino-bis (3-ethylbenzothiazloine-6-sulfonic acid) radical cation (ABTS), ferric reducing antioxidant power (FRAP), antimicrobial and inhibitory properties against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and porcine pancreatic lipase (PPL). The quality of the irrigation water did not affect the essential oil yield quantity and quality demonstrating the potential of this marginal water source for agri-industrial production of OSS. Under both irrigation water qualities, the EO contained 35 constituents, of which thymol (40.4–46.7%), and carvacrol (35.1–41.8%) were the most abundant. EOs from plants of both irrigation water qualities exhibited a strong free radical scavenging ability and reducing power activity, and high AChE, BChE, and PPL inhibitory activities. The EOs also showed a moderate antibacterial activity against most test bacterial strains, with high antibacterial activity against Staphylococcus aureus with 117 percent inhibition compared with chloramphenicol (30μg/disc). The high inhibitory activity of the EOs on AChE, BChE, and PPL, and its high antioxidant and antibacterial activities may be due to high concentrations of monoterpenes, especially thymol and carvacrol. The data demonstrate that secondary-treated municipal effluents can be successfully utilized for irrigation of industrial crop production setups for essential oil production from OSS (as a source of natural antioxidants, anticholinesterase, antiobesity and antistaphylococcal agents) without compromising yield quantity and bioactivities compared with potable water irrigation.