Anticoagulant rodenticides (ARs) are widely used for pest control, resulting in their pervasive presence in the environment and posing significant toxicological risks to a range of predatory and scavenging species. Our study mainly aimed to evaluate AR exposure and effects in nestlings of eagle owl (Bubo bubo) from the Region of Murcia (southeastern Spain). We analysed ARs in blood samples (n = 106) using high-performance liquid chromatography-triple quadrupole (HPLC-TQ), assessed the influence of potential anthropogenic (presence of livestock farms, landfills and human population density) and environmental (land uses and proximity to watercourses) variables, and measured prothrombin time (PT) and plasma biochemical parameters as biomarkers of effects. Our results showed the presence of AR residues in 91.5% of the nestlings, with 70.8% exhibiting multiple ARs (up to six compounds in a single individual). Second-generation ARs (SGARs) were the most prevalent compounds. The analysis of biochemical parameters indicated that the sampled individuals were in good physiological condition. Although PT was positively correlated with total AR concentration (Sigma ARs), the relationship was not significant (Rho = 0.04; p = 0.49). Regarding environmental factors, higher Sigma ARs were associated with the most urbanised study site and the presence of landfills, likely due to the increased availability of rodent prey. The prevalence of two SGARs (brodifacoum and difenacoum) was linked to closer proximity to riverbeds, suggesting a contamination pathway associated with inland aquatic ecosystems, where these AR compounds may concentrate due to water scarcity. This study underscores the widespread exposure of eagle owls to ARs and highlights the importance of effective monitoring and management of these pollutants to protect conservation-concern wildlife in Mediterranean semiarid regions.