Due to the high environmental risks of chemical pesticides, biological control of plant pests with bio-pesticides is highly encouraged and recommended. Chitosan and oligochitosan are well-known biological control agents for their nontoxic, biodegradable, and biocompatible properties. A field experiment was conducted at Nubaria Research Station during the 2020/2021 and 2021/2022 growing seasons to study the effect of different applications of chitosan on barley plants against aphid infestation under field artificial infestation conditions as well as yield and physiologic related traits. The experiment was laid out in a randomized complete block design (RCBD) with three replications and seven treatments among spraying, soaking, and combinations between them at two concentrations 250 and 500 mg. The study results revealed that there were significant variations among the seven treatments for all studied traits. The highest mortality percent was obtained from the treatment T7 (soaking seeds & spraying with 500 mg/L.), which was 99.5 % compared with untreated 2.66%; this treatment induced an increase in the thickness of grains and stem tissues, which caused a reduction in the feeding of aphids, resulting in a clear drop in population number. Significant increases were recorded for total phenolic content, protein content, yield and its components in T7 and T5 treatments.