Water safety and security has become a great problem in recent years, as the shortage of water resources in most countries is becoming an increasingly serious issue. Therefore, searching for new water sources or treating contaminated water has become a major requirement to covering the shortage of fresh water. The current study evaluated the impact of Trichoderma asperellum on the physicochemical characterization of agricultural run-off (AR) compared with groundwater (GW). Chemical oxygen demand (COD), biological oxygen demand (BOD5), ammonia, total nitrogen (TN), and total dissolved salts (TDS) of AR were higher than that of GW with 90, 90.2, 91.9, 78, and 61.6%, respectively. The addition of T. asperellum to AR caused the COD, BOD5, ammonia, TN, and TDS to decrease 47.2, 39.2, 16.2, 12.9, and 4.8%, respectively. Contents of metals in AR or GW were decreased by T. asperellum. Microbial analysis of soil irrigated by AR in contrast to irrigated by GW showed the presence of a high number of bacteria (32 × 106 CFU), actinomycetes (18 × 104 CFU), and fungi (13 × 104 CFU). Trifolium alexandrinum growth and its antioxidant enzymes activities (catalase, superoxide dismutase, and ascorbate peroxidase) were enhanced by AR inoculated by T. asperellum compared with un-inoculated AR or GW.