Abstract: Background and Objective: The use of antibiotics in animal production is a major concern to health providers and consumers. This study was carried out to determine the prevalence of resistant Escherichia coli in cow milk and hands of milkers. Materials and Methods: A total of 104 cow milk and hands of milkers were examined for the presence of Escherichia coli. Isolation of Escherichia coli was done using the convention method in the US Food and Drug Administration-Bacteriological Analysis Manual (FDA-BAM). Antibiotic susceptibility test was done using the disc diffusion method and the results interpreted using the clinical and laboratory standards institute guidelines. Prevalence data was analyzed using SPSS version 17. Results: The overall prevalence of Escherichia coli in the milk and hand samples was 40.38%. The prevalence of Escherichia coli in milk collected directly from the udder, in milk collected from milking containers, right hand swabs and left hand swabs were 61.54, 57.69, 23.08 and 19.23%, respectively. The prevalence of Escherichia coli in milk samples was significantly higher (p<0.05) than hand samples. Twenty seven Escherichia coli isolated from the milk and hand samples were screened against 8 different antibiotics. Overall, 14.35% of the Escherichia coli isolates were resistant, 21.30% were intermediate and 64.35% were susceptible. Resistance to ceftriaxone (29.63%) was the highest, followed by tetracycline (25.93%) and ampicillin (22.22%). A relatively higher percentage of the isolates exhibited intermediate resistance to ampicillin (51.85%), erythromycin (48.15%) and chloramphenicol (37.04%). Escherichia coli isolates also exhibited 13 antibiotic resistant patterns. Five isolates were resistant to 3 or more different classes of antibiotics. Conclusion: This study revealed that Escherichia coli from cow milk and hands of milkers in the Nyankpala community are resistant to some antibiotics. Consumers are expose to Escherichia coli infection from drinking of milk produced in the Nyankpala community of Ghana.