Aim: This study’s aim was to assess the knowledge, attitudes, and practices of mothers regarding childhood vaccination during the first five years of children’s lives in Saudi Arabia. Method: A cross-sectional, descriptive study was conducted with the application of convenience sampling, and 262 questionnaires were completed by Saudi mothers who had children aged 5 years or younger. Results: The majority of the participants were aged 25–31 years (57%), 61% held a bachelor’s degree, and 60.3% had children aged 2–5 years. The knowledge score was 86%, 2492 out of a total score of 2893; the attitude score was 89.1%, 973 out of a total score of 1052; the practice score was 80.5%, 1059 out of a total score of 1315. There was no evidence of an association (p > 0.05) between the knowledge, attitudes, and practice of mothers regarding vaccination and their sociodemographic aspects. Conclusion: The Saudi mothers in our sample were knowledgeable, with positive attitudes regarding vaccination, and they demonstrated good practices. This might be explained by the higher educational level of our sample. Recommendations: We recommend using multiple educational methods to support the practice of mothers regarding the management of complications of vaccinations.