Past studies have shown that healthcare professionals may lack awareness and knowledge regarding oxygen therapy, and its implementation often has several obstacles. This study was carried out to investigate the effect of an educational program about oxygen therapy on nurses' knowledge and practices. This cross-sectional, quasi-experimental study was conducted in 2022 at the pediatric department of Nishtar Hospital, Multan, where 160 nurses from primary and secondary health centers attended an educational program delivered in the pediatric department. The pre-test-post-test approach was used to evaluate the effectiveness of the structured educational program. The independent variable was the educational program, and the dependent variable was the nurses' knowledge and practice about oxygen toxicity. Data analysis was performed using SPSS version 23 (IBM Corp., New York, USA). The data were tabulated as means and standard deviations for numerical values and frequency percentages for categorical values. The student's -test and the chi-square test were applied to investigate any associations among variables. The average test scores before and after the implementation of the educational program were 10.75±2.65 and 17.52±2.04, respectively. The average post-test score was greater than that of the pre-test, and the difference was statistically significant (p<0.001). The study found that after the implementation of the educational program, the knowledge and practices of nurses regarding oxygen therapy improved significantly, with the majority showing a positive attitude toward the program.