Exposure to arsenic (As) can induce numerous lethal diseases, such as cancer, cardiovascular issues, skin diseases, and diabetes in humans. The major route of human and animal exposure to As is through drinking As-rich groundwater. This study assessed As occurrence in the groundwater of two districts in the Punjab (Vehari and Lodhran) provinces of Pakistan. Groundwater analysis revealed an average As concentration of 7.7 µg/L (n = 79) in the study area, with a maximum As concentration up to 41.4 µg/L (33% of samples exceeding the WHO limit of 10 µg/L). Arsenic traces were found in animal milk (n = 15, mean: 0.79 µg/L, 17% exceeding 2.0 µg/L), human hair (n = 12, mean: 0.36 µg/g, 17% exceeding 1.0 µg/g), and human nails (n = 8, mean: 0.03 µg/g, none of the samples exceeded 1.0 µg/g). Health risk assessment indices revealed that about 33% of the hazard quotient and 54% of the cancer risk factor exceeded their thresholds. Despite the low–moderate As concentration in groundwater and the accumulation of As in a few biological samples, there is a possibility of potential As poisoning via the long-term and continuous use of groundwater for drinking. Monitoring and blanket testing of wells for As in well water can provide baseline data to minimize the threat of As-mediated arsenicosis in As-affected areas of Pakistan. Moreover, a detailed study of potential As accumulation in biological samples with a higher number of samples is recommended in the area.