Background: Conjunctivitis is a frequent infection, particularly in young people. Conjunctivitis is highly contagious and uncomfortable, but it seldom causes permanent vision loss or structural damage. This study aimed to evaluate the effect of instructional guidelines on mothers' knowledge and reported practices regarding Conjunctivitis among Children in Ophthalmology outpatient clinic. Design: A pretest/post-test quasi-experimental research design was used. Setting: This study was conducted at the Ophthalmology outpatient clinics at Sohag University Hospital. Sample: The study participants included a convenient sample of 100 mothers. Tools used for data collection: Tool (1): A structured interview questionnaire which consisted of three parts: (I) children's demographic data; (II) mothers demographic data, (III) children's medical history of conjunctivitis; Tool (2): Mothers' knowledge assessment sheet about conjunctivitis, Tool (3): Mothers' reported practices assessment sheet. Results: The results revealed that highly statistically significant differences were found between mothers' knowledge and reported practices regarding Conjunctivitis pre and post-instructional guidelines implementation. Conclusion The current study found that the mother's knowledge and reported practices had improved as a result of the instructional guidelines implementation. Recommendations The ophthalmology outpatient clinics at Sohag University Hospital should hold mother education seminars and programs regarding the treatment and prevention of conjunctivitis and associated infection.