Introduction: Nowadays, infertility represents a multifaceted health issue that significantly impacts numerous couples, giving rise to notable psychological and social complexities. Notably, one in six couples experience infertility, with approximately 50% of cases attributed to male factors. Male infertility, a complex disorder influenced by both environmental factors and genetic predisposition, involves the interplay of various genes contributing to its manifestation. It can be categorized into specific subtypes, including asthenozoospermia. The primary aim of this study was to identify novel variants associated with asthenozoospermia within the Greek population and to elucidate the roles of the genes involved. Materials and Methods: Whole-genome sequencing (WGS) was conducted on both normozoospermic and asthenozoospermic individuals. Following the identification of variants exclusively present in asthenozoospermic men, an extensive range of tools, functional assessments, and predictive algorithms were employed to prioritize these variants. Results: The investigation unveiled numerous polymorphisms, comprising 155 classified as high-impact and 715 classified as moderate-impact. While several of these variants were found within genes previously linked to male infertility, a notable subset was associated with asthenozoospermia for the first time. Furthermore, pathway enrichment analysis and Gene ontology (GO) analyses revealed polymorphisms on genes implicated in teratozoospermia through various mechanisms and pathways. Conclusions: This study reaffirms the involvement of previously studied genes in male infertility, while also shedding light on novel molecular mechanisms. By providing a comprehensive list of variants and candidate genes associated with asthenozoospermia within the Greek population, this research contributes significantly to our understanding of male infertility and paves the road for future studies.