In order to evaluation the quantity and quality of thyme (Thymus vulgaris L.) essential oil under different irrigation levels and separate and integrative application of biological fertilizer and chitosan nano fertilizer, a field experiment was carried out as a split plot based on a randomized complete block design (RCBD) with 12 treatments and three replications at the faculty of Agriculture, University of Maragheh, Iran. The main factor was different irrigation levels contain irrigation at 80% field capacity (FC80) as no stress, 60%FC as moderate water stress (FC60) and 40% FC as severe water stress (FC40). Also, the sub-factor was four different fertilizer sources including non-application of fertilizer (control), application of arbuscular mycorrhizal fungi (AMF), chitosan NPs (CHT) and AMF+CHT NPs. The results demonstrated that the dry yield of thyme decreased by 13 and 40.3% under FC60 and FC40 water stress conditions. However, co-application of AMF+CHT NPs enhanced the dry yield of thyme by 21.7% in comparison with control (non-application of fertilizer). The maximum EO content (2.03%) and EO yield (10.04 g m-2) of thyme was obtained under moderate water stress (FC60) fertilized with AMF+CHT NPs. Co-application of AMF+CHT NPs enhanced the EO con-tent and EO yield of thyme by 17.1 and 42.7%, respectively. Based on the analysis of essential oil compounds, the highest content of main essential oil constituents of thyme such as thymol and -terpinene was obtained under moderate water stress (FC60) fertilized with AMF+CHT NPs. In addition, the highest content of p-cymene and carvacrol was observed in the severe water stress (FC40) fertilized with AMF+CHT NPs. The present research suggests that co-application of AMF+CHT NPs represent a sustainable and eco-friendly strategy for improving the EO quantity and quality under water stress conditions.