Purpose This study aimed to assess knowledge and attitude towards the human papillomavirus vaccine and associated factors among mothers who have eligible daughters. Methods A community-based cross-sectional study with a total sample of 601 study participants was conducted in Debre Markos town from February to March 2021. The study participants were selected using a multistage sampling technique from mothers who have eligible daughters. The data was collected using an interview administration questionnaire. The data were entered into EPI data version 3.1 then, exported to SPSS version 25 for analysis. Variables with a p-value of < 0.25 in bivariate were a candidate for multivariate and those variables with a p-value of <0.05 in multivariate binary logistic regression were considered as a statistically significant factor for knowledge and attitude of the HPV vaccine. Results A total of 601 study participants with a response rate of 100% were included in the study. Among participants in this study, 47.6% have good knowledge and 77.4% have a positive attitude towards the HPV vaccine. Knowledge of mothers about HPV vaccine was affected by having degree and above (adjusted odds ratio (AOR) =7.687; 95% CI=1.837–32.168; p=0.005), did not hear about HPV vaccine (AOR=0.172; 95% CI=0.098–0.302; P=0.000), and positive attitude towards HPV vaccine (AOR=2.959; 95% CI=1.580–5.539; P=0.001). Attitude of mothers towards HPV vaccine was affected by did not hear about HPV vaccine (AOR=0.285; 95% CI=0.163–0.499; P=000), and good knowledge about HPV vaccine (AOR=2.705; 95% CI=1.454–5.035; 0.002). Conclusion Knowledge of mothers who have eligible daughters about HPV vaccine was low. Maternal educational level, hearing about the HPV vaccine, and attitude towards HPV vaccine were significantly associated predictors with knowledge towards HPV vaccine. However, the attitude was high and factors associated with attitudes towards the HPV vaccine were knowledge about the HPV vaccine and hearing about the HPV vaccine. An integrated work should be considered by Debre Markos town health office with other non-governmental organizations working on health and health-related issues to improve knowledge about the importance of HPV vaccine for mothers who have eligible daughters.