This study aimed to investigate the different effects of fatigue and health-related quality of life in the Turkish mothers of children with autism spectrum disorder (ASD) through comparisons with mothers of typically developing children, and to evaluate the relationship between different aspects of maternal fatigue, depression, and maternal health-related quality of life. The study included a total of 103 mothers, comprising 60 mothers of children with ASD and 43 mothers of typically developing children. The Nottingham Health Profile and Quality of Life in Autism Questionnaire-Parent Version were used to assess the health-related quality of life. Fatigue was assessed comprehensively with the Fatigue Impact Scale and the Fatigue Severity Scale. The Nottingham Health Profile total and physical mobility domain and the quality of life were the strongest factors, explaining with a variance of 66.7% the fatigue impact on cognitive function. The mothers’ quality of life accounted for 64.8% of the variance in factors that explained fatigue. Physical mobility, social isolation and quality of life were associated with the physical impact of fatigue and were explained with a variance of 52.4%. Emotional reactions and quality of life were explained with a variance of 52.7% in the fatigue of psychosocial function. The current study has highlighted that the fatigue of Turkish mothers of children with ASD has a greater impact on cognitive, physical and psychosocial functions. Furthermore, the quality of life, physical mobility, social isolation, and emotional reactions of mothers have a significant impact on maternal fatigue.