Cancer patients face multiple challenges, such as infertility which may result from exposure to irradiation during cancer treatment. Little is known about the health practitioners’ knowledge and practice regarding fertility preservation and its available options in Saudi Arabia. This study aimed to assess healthcare practitioners’ knowledge, attitudes, and practices (KAP) toward fertility preservation among cancer patients in Makkah, Sau-di Arabia. A cross-sectional study was conducted among 100 health practitioners from September 2022 to January 2023. A self-administered questionnaire was used to assess KAP. The Chi-square (χ ² ) test and Student’s-t-test were used for categorical data and continuous variables as appropriate. Most participants (90%) lacked knowledge about fertility preservation. Cost and clinic availability significantly influenced the health practitioners’ attitude toward fertility preservation discussions with cancer patients (P < 0.05). Most of the study participants (87%) were familiar with sperm and egg freezing, while other techniques were less well-known. There were significant associations between health practitioners’ attitudes in discussing fertility preservation with their cancer patients with significant influence (P <0.05). The results revealed that 92% of the participants agreed that the Saudi Ministry of Health should establish practice guidelines and provide fertility preservation services for cancer patients. Healthcare practitioners in Makkah, Saudi Arabia, have limited knowledge about fertility preservation. Educational interventions and improved access to fertility preservation services are needed.